Combinations of AKT inhibitor compounds and erlotinib, and methods of use

ABSTRACT

The invention provides A combination of, a) a compound of Formula Ia: 
                         
or a pharmaceutically acceptable salt thereof; and b) erlotinib or a pharmaceutically acceptable salt thereof for the prophylactic or therapeutic treatment of a hyperproliferative disorder, such as cancer.

PRIORITY OF INVENTION

This application is a continuation of U.S. patent application Ser. No.14/009,313, filed Oct. 1, 2013, which is a 35 U.S.C. §371 application ofInternational Application No. PCT/US2012/031671, filed Mar. 30, 2012,which claims the benefit of U.S. Provisional Application No. 61/470,803,filed Apr. 1, 2011, and U.S. Provisional Application No. 61/470,624,filed Apr. 1, 2011. The entire content of the applications referencedabove are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates generally to pharmaceutical combinations ofcompounds with activity against hyperproliferative disorders such ascancer and which include compounds that inhibit AKT kinase activity. Theinvention also relates to methods of using the combinations for invitro, in situ, and in vivo diagnosis or treatment of mammalian cells,or associated pathological conditions.

BACKGROUND OF THE INVENTION

Protein kinases (PK) are enzymes that catalyze the phosphorylation ofhydroxy groups on tyrosine, serine and threonine residues of proteins bytransfer of the terminal (gamma) phosphate from ATP. Through signaltransduction pathways, these enzymes modulate cell growth,differentiation and proliferation, i.e., virtually all aspects of celllife in one way or another depend on PK activity (Hardie, G. and Hanks,S. (1995) The Protein Kinase Facts Book. I and II, Academic Press, SanDiego, Calif.). Furthermore, abnormal PK activity has been related to ahost of disorders, ranging from relatively non-life threatening diseasessuch as psoriasis to extremely virulent diseases such as glioblastoma(brain cancer). Protein kinases are an important target class fortherapeutic modulation (Cohen, P. (2002) Nature Rev. Drug Discovery1:309).

International Patent Application Publication Number WO 2008/006040discusses a series of inhibitors of AKT of formula I:

Currently, there remains a need for improved methods and compositionsthat can be used to treat hyperproliferative diseases such as cancer.

SUMMARY OF THE INVENTION

It has been determined that additive or synergistic effects ininhibiting the growth of cancer cells in vitro and in vivo can beachieved by administering a compound of formula I or a pharmaceuticallyacceptable salt thereof in combination with certain other specificchemotherapeutic agents. The combinations and methods may be useful inthe treatment of hyperproliferative disorders such as cancer.

One aspect of the invention provides a method for treating ahyperproliferative disorder in a mammal comprising, administering to themammal, a) a compound of formula I:

or a pharmaceutically acceptable salt thereof; and b) one or more agentsselected from 5-FU, a platinum agent (carboplatin, cisplatnin,oxaliplatin, etc.) irinotecan, docetaxel, doxorubicin, gemcitabine,SN-38, capecitabine, temozolomide, erlotinib, PD-0325901, paclitaxel,bevacizumab, pertuzumab, tamoxifen, rapamycin, lapatinib, PLX-4032,MDV3100, abiraterone, and GDC-0973.

The compound of formula I or the pharmaceutically acceptable saltthereof and the chemotherapeutic agent may be co-formulated foradministration in a combination as a pharmaceutical composition or theymay be administered separately in alternation (sequentially) as atherapeutic combination.

One aspect of the invention provides a method for treating a disease orcondition modulated by AKT kinase in a mammal comprising, administeringto the mammal, a) a compound of formula I or a pharmaceuticallyacceptable salt thereof; and b) one or more agents selected from 5-FU, aplatinum agent, irinotecan, docetaxel, doxorubicin, gemcitabine, SN-38,capecitabine, temozolomide, erlotinib, PD-0325901, paclitaxel,bevacizumab, pertuzumab, tamoxifen, rapamycin, lapatinib, PLX-4032,MDV3100, abiraterone, and GDC-0973.

One aspect of the invention provides the combination of a) a compound offormula I or a pharmaceutically acceptable salt thereof; and b) one ormore agents selected from 5-FU, a platinum agent, irinotecan, docetaxel,doxorubicin, gemcitabine, SN-38, capecitabine, temozolomide, erlotinib,PD-0325901, paclitaxel, bevacizumab, pertuzumab, tamoxifen, rapamycin,lapatinib, PLX-4032, MDV3100, abiraterone, and GDC-0973 for treating ahyperproliferative disorder.

One aspect of the invention provides the combination of a) a compound offormula I or a pharmaceutically acceptable salt thereof; and b) one ormore agents selected from 5-FU, a platinum agent, irinotecan, docetaxel,doxorubicin, gemcitabine, SN-38, capecitabine, temozolomide, erlotinib,PD-0325901, paclitaxel, bevacizumab, pertuzumab, tamoxifen, rapamycin,lapatinib, PLX-4032, MDV3100, abiraterone, and GDC-0973 for treating adisease or condition modulated by AKT kinase.

One aspect of the invention provides the use of a compound of formula Ior a pharmaceutically acceptable salt thereof in the preparation of amedicament for the treatment of a hyperproliferative disorder in amammal, wherein one or more agents selected from 5-FU, a platinum agent,irinotecan, docetaxel, doxorubicin, gemcitabine, SN-38, capecitabine,temozolomide, erlotinib, PD-0325901, paclitaxel, bevacizumab,pertuzumab, tamoxifen, rapamycin, lapatinib, PLX-4032, MDV3100,abiraterone, and GDC-0973 are administered to the mammal.

One aspect of the invention provides the use of a compound of formula Ior a pharmaceutically acceptable salt thereof in the preparation of amedicament for the treatment of a disease or condition modulated by AKTkinase in a mammal, wherein one or more agents selected from 5-FU, aplatinum agent, irinotecan, docetaxel, doxorubicin, gemcitabine, SN-38,capecitabine, temozolomide, erlotinib, PD-0325901, paclitaxel,bevacizumab, pertuzumab, tamoxifen, rapamycin, lapatinib, PLX-4032,MDV3100, abiraterone, and GDC-0973 are administered to the mammal.

One aspect of the invention provides a kit comprising a compound offormula I or a pharmaceutically acceptable salt thereof, a container,and a package insert or label indicating the administration of thecompound of formula I or a pharmaceutically acceptable salt thereof withone or more agents selected from 5-FU, a platinum agent, irinotecan,docetaxel, doxorubicin, gemcitabine, SN-38, capecitabine, temozolomide,erlotinib, PD-0325901, paclitaxel, bevacizumab, pertuzumab, tamoxifen,rapamycin, lapatinib, PLX-4032, MDV3100, abiraterone, and GDC-0973 fortreating a hyperproliferative disorder.

One aspect of the invention provides a product comprising a compoundhaving formula I or a pharmaceutically acceptable salt thereof, and achemotherapeutic agent selected from 5-FU, a platinum agent, irinotecan,docetaxel, doxorubicin, gemcitabine, SN-38, capecitabine, temozolomide,erlotinib, PD-0325901, paclitaxel, bevacizumab, pertuzumab, tamoxifen,rapamycin, lapatinib, PLX-4032, MDV3100, abiraterone, and GDC-0973; as acombined preparation for separate, simultaneous or sequential use in thetreatment of a hyperproliferative disorder.

In addition to providing improved treatment for a givenhyperproliferative disorder, administration of certain combinations ofthe invention may improve the quality of life for a patient compared tothe quality of life experienced by the same patient receiving adifferent treatment. For example, administration of a combination of acompound of formula I or a pharmaceutically acceptable salt thereof, anda chemotherapeutic agent as described herein to a patient may provide animproved quality of life compared to the quality of life the samepatient would experience if they received only the chemotherapeuticagent as therapy. For example, the combined therapy with the combinationdescribed herein may lower the dose of chemo agents needed, therebylessening the side-effects associated with high-dose chemotherapeuticagents (e.g., nausea, vomiting, hair loss, rash, decreased appetite,weight loss, etc.). The combination may also cause reduced tumor burdenand the associated adverse events, such as pain, organ dysfunction,weight loss, etc. Accordingly, one aspect of the invention provides acompound of formula I or a pharmaceutically acceptable salt thereof, fortherapeutic use for improving the quality of life of a patient treatedfor a hyperproliferative disorder with an agent selected from 5-FU, aplatinum agent, irinotecan, docetaxel, doxorubicin, gemcitabine, SN-38,capecitabine, temozolomide, erlotinib, PD-0325901, paclitaxel,bevacizumab, pertuzumab, tamoxifen, rapamycin, lapatinib, PLX-4032,MDV3100, abiraterone, and GDC-0973. Accordingly, another aspect of theinvention provides a compound of formula I or a pharmaceuticallyacceptable salt thereof, for therapeutic use for improving the qualityof life of a patient treated for a hyperproliferative disorder witherlotinib.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show data for the compound of Formula 1a (GDC-0068) anderlotinib in NCI-H2122 NSCLC tumors.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS AND DEFINITIONS

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and claims are intended tospecify the presence of stated features, integers, components, or steps,but they do not preclude the presence or addition of one or more otherfeatures, integers, components, steps, or groups thereof.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms, wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. Examples ofalkyl groups include, but are not limited to, methyl (Me, —CH₃), ethyl(Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to twelve carbon atoms with at least one siteof unsaturation, i.e., a carbon-carbon, sp² double bond, wherein thealkenyl radical may be optionally substituted independently with one ormore substituents described herein, and includes radicals having “cis”and “trans” orientations, or alternatively, “E” and “Z” orientations.Examples include, but are not limited to, ethylenyl or vinyl (—CH═CH₂),allyl (—CH₂CH═CH₂), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to twelve carbon atoms with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms as a monocyclic ring or 7to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to12 atoms can be arranged, for example, as a bicyclo[4,5], [5,5], [5,6]or [6,6] system, and bicyclic carbocycles having 9 or 10 ring atoms canbe arranged as a bicyclo[5,6] or [6,6] system, or as bridged systemssuch as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Some aryl groups are representedin the exemplary structures as “Ar”. Aryl includes bicyclic radicalscomprising an aromatic ring fused to a saturated, partially unsaturatedring, or aromatic carbocyclic or heterocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.Aryl groups are optionally substituted independently with one or moresubstituents described herein.

The terms “heterocycle,” “hetercyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 20 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen and sulfur,the remaining ring atoms being C, where one or more ring atoms isoptionally substituted independently with one or more substituentsdescribed below. A heterocycle may be a monocycle having 3 to 7 ringmembers (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O,P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atomsand 1 to 6 heteroatoms selected from N, O, P, and S), for example: abicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles are describedin Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and9; “The Chemistry of Heterocyclic Compounds, A series of Monographs”(John Wiley & Sons, New York, 1950 to present), in particular Volumes13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. The term“heterocycle” includes heterocycloalkoxy. “Heterocyclyl” also includesradicals where heterocycle radicals are fused with a saturated,partially unsaturated ring, or aromatic carbocyclic or heterocyclicring. Examples of heterocyclic rings include, but are not limited to,pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino,morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl,azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl,oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinylimidazolinyl, imidazolidinyl,3-azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,azabicyclo[2.2.2]hexanyl, 3H-indolyl quinolizinyl and N-pyridyl ureas.Spiro moieties are also included within the scope of this definition.Examples of a heterocyclic group wherein 2 ring carbon atoms aresubstituted with oxo (═O) moieties are pyrimidinonyl and1,1-dioxo-thiomorpholinyl. The heterocycle groups herein are optionallysubstituted independently with one or more substituents describedherein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl,benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.Heteroaryl groups are optionally substituted independently with one ormore substituents described herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked),nitrogen (nitrogen-linked) or oxygen (oxygen-linked) attached where suchis possible. By way of example and not limitation, carbon bondedheterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6 of apyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4,or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole ortetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole orthiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole,position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine,position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5,6, 7, or 8 of an isoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the growth, development or spread of cancer. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) prevents or delays the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer. Gastriccancer, as used herein, includes stomach cancer, which can develop inany part of the stomach and may spread throughout the stomach and toother organs; particularly the esophagus, lungs, lymph nodes, and theliver.

A “chemotherapeutic agent” is a biological (large molecule) or chemical(small molecule) compound useful in the treatment of cancer, regardlessof mechanism of action. Classes of chemotherapeutic agents include, butare not limited to: alkylating agents, antimetabolites, spindle poisonplant alkaloids, cytotoxic/antitumor antibiotics, topoisomeraseinhibitors, proteins, antibodies, photosensitizers, and kinaseinhibitors. Chemotherapeutic agents include compounds used in “targetedtherapy” and non-targeted conventional chemotherapy.

The term “mammal” includes, but is not limited to, humans, mice, rats,guinea pigs, monkeys, dogs, cats, horses, cows, pigs, sheep, andpoultry.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound is a base, the desired pharmaceutically acceptable saltmay be prepared by any suitable method available in the art, forexample, treatment of the free base with an inorganic acid, such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.Acids which are generally considered suitable for the formation ofpharmaceutically useful or acceptable salts from basic pharmaceuticalcompounds are discussed, for example, by P. Stahl et al, Camille G.(eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use.(2002) Zurich: Wiley-VCH; S. Berge et al, Journal of PharmaceuticalSciences (1977) 66(1) 1 19; P. Gould, International J. of Pharmaceutics(1986) 33 201 217; Anderson et al, The Practice of Medicinal Chemistry(1996), Academic Press, New York; Remington's Pharmaceutical Sciences,18^(th) ed., (1995) Mack Publishing Co., Easton Pa.; and in The OrangeBook (Food & Drug Administration, Washington, D.C. on their website).These disclosures are incorporated herein by reference thereto.

If the compound is an acid, the desired pharmaceutically acceptable saltmay be prepared by any suitable method, for example, treatment of thefree acid with an inorganic or organic base, such as an amine (primary,secondary or tertiary), an alkali metal hydroxide or alkaline earthmetal hydroxide, or the like. Illustrative examples of suitable saltsinclude, but are not limited to, organic salts derived from amino acids,such as glycine and arginine, ammonia, primary, secondary, and tertiaryamines, and cyclic amines, such as piperidine, morpholine andpiperazine, and inorganic salts derived from sodium, calcium, potassium,magnesium, manganese, iron, copper, zinc, aluminum and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

A “solvate” refers to a physical association or complex of one or moresolvent molecules and a compound of the invention. The compounds mayexist in unsolvated as well as solvated forms. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.The term “hydrate” refers to the complex where the solvent molecule iswater. This physical association involves varying degrees of ionic andcovalent bonding, including hydrogen bonding. In certain instances thesolvate will be capable of isolation, for example when one or moresolvent molecules are incorporated in the crystal lattice of thecrystalline solid. Preparation of solvates is generally known, forexample, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601 611 (2004).Similar preparations of solvates, hemisolvate, hydrates and the like aredescribed by E. C. van Tonder et al, AAPS PharmSciTech., 5(1), article12 (2004); and A. L. Bingham et al, Chem. Commun., 603 604 (2001). Atypical, non-limiting, process involves dissolving the inventivecompound in desired amounts of the desired solvent (organic or water ormixtures thereof) at a higher than ambient temperature, and cooling thesolution at a rate sufficient to form crystals which are then isolatedby standard methods. Analytical techniques such as, for example I.R.spectroscopy, show the presence of the solvent (or water) in thecrystals as a solvate (or hydrate).

The term “synergistic” as used herein refers to a therapeuticcombination which is more effective than the additive effects of the twoor more single agents. A determination of a synergistic interactionbetween a compound of formula I or a pharmaceutically acceptable saltthereof and one or more chemotherapeutic agent may be based on theresults obtained from the assays described herein. The results of theseassays can be analyzed using the Chou and Talalay combination method andDose-Effect Analysis with CalcuSyn software in order to obtain aCombination Index (Chou and Talalay, 1984, Adv. Enzyme Regul. 22:27-55).The combinations provided by this invention have been evaluated inseveral assay systems, and the data can be analyzed utilizing a standardprogram for quantifying synergism, additivism, and antagonism amonganticancer agents. The program utilized is that described by Chou andTalalay, in “New Avenues in Developmental Cancer Chemotherapy,” AcademicPress, 1987, Chapter 2. Combination Index values less than 0.8 indicatessynergy, values greater than 1.2 indicate antagonism and values between0.8 to 1.2 indicate additive effects. The combination therapy mayprovide “synergy” and prove “synergistic”, i.e., the effect achievedwhen the active ingredients used together is greater than the sum of theeffects that results from using the compounds separately. A synergisticeffect may be attained when the active ingredients are: (1)co-formulated and administered or delivered simultaneously in acombined, unit dosage formulation; (2) delivered by alternation or inparallel as separate formulations; or (3) by some other regimen. Whendelivered in alternation therapy, a synergistic effect may be attainedwhen the compounds are administered or delivered sequentially, e.g., bydifferent injections in separate syringes. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together. In some examples, Combination effects wereevaluated using both the BLISS independence model and the highest singleagent (HSA) model (Lehár et al. 2007, Molecular Systems Biology 3:80).BLISS scores quantify degree of potentiation from single agents and aBLISS score>0 suggests greater than simple additivity. An HSA score>0suggests a combination effect greater than the maximum of the singleagent responses at corresponding concentrations.

In one aspect the invention provides a method for treating thehyperproliferative disorder wherein administration of the compound offormula I or the salt thereof and the one or more agents selected from5-FU, a platinum agent, irinotecan, docetaxel, doxorubicin, gemcitabine,SN-38, capecitabine, temozolomide, erlotinib, PD-0325901, paclitaxel,bevacizumab, pertuzumab, tamoxifen, rapamycin, lapatinib, PLX-4032,MDV3100, abiraterone, and GDC-0973 provides a synergistic effect intreating the hyperproliferative disorder. In a further aspect, thesynergistic effect has a Combination Index value of less than about 0.8.In another aspect the invention provides a method for treating thehyperproliferative disorder wherein administration of the compound offormula I or the salt thereof and the one or more agents selected fromerlotinib provides a synergistic effect in treating thehyperproliferative disorder. In a further aspect, the synergistic effecthas a Combination Index value of less than about 0.8.

Formula I Compounds

Formula I compounds include a compound of formula I:

and pharmaceutically acceptable salts thereof, wherein:

R¹ is H, Me, Et, vinyl, CF₃, CHF₂ or CH₂F;

R² is H or Me;

R⁵ is H, Me, Et, or CF₃;

A is

G is phenyl optionally substituted by one to four R⁹ groups or a 5-6membered heteroaryl optionally substituted by a halogen;

R⁶ and R⁷ are independently H, OCH₃, (C₃-C₆ cycloalkyl)-(CH₂), (C₃-C₆cycloalkyl)-(CH₂CH₂), V—(CH₂)₀₋₁ wherein V is a 5-6 membered heteroarylhaving from one to two ring heteroatoms independently selected from N, Oand S, W—(CH₂)₁₋₂ wherein W is phenyl optionally substituted with F, Cl,Br, I, OMe, CF₃ or Me, C₃-C₆-cycloalkyl optionally substituted withC₁-C₃ alkyl or O(C₁-C₃ alkyl), hydroxy-(C₃-C₆-cycloalkyl),fluoro-(C₃-C₆-cycloalkyl), CH(CH₃)CH(OH)phenyl, 4-6 membered heterocycleoptionally substituted with F, OH, C₁-C₃-alkyl, cyclopropylmethyl orC(═O)(C₁-C₃ alkyl), or C₁-C₆-alkyl optionally substituted with one ormore groups independently selected from OH, oxo, O(C₁-C₆-alkyl), CN, F,NH₂, NH(C₁-C₆-alkyl), N(C₁-C₆-alkyl)₂, cyclopropyl, phenyl, imidazolyl,piperidinyl, pyrrolidinyl, morpholinyl, tetrahydrofuranyl, oxetanyl, ortetrahydropyranyl, or R⁶ and R⁷ together with the nitrogen to which theyare attached form a 4-7 membered heterocyclic ring, wherein saidheterocyclic ring is optionally substituted with one or more groupsindependently selected from OH, halogen, oxo, CF₃, CH₂CF₃, CH₂CH₂OH,O(C₁-C₃ alkyl), C(═O)CH₃, NH₂, NHMe, N(Me)₂, S(O)₂CH₃, cyclopropylmethyland C₁-C₃ alkyl;

R^(a) and R^(b) are H,

or R^(a) is H, and R^(b) and R⁶ together with the atoms to which theyare attached form a 5-6 membered heterocyclic ring having one or tworing nitrogen atoms;

R^(c) and R^(d) are H or Me,

or R^(c) and R^(d) together with the atom to which they are attachedfrom a cyclopropyl ring;

R⁸ is H, Me, F or OH,

or R⁸ and R⁶ together with the atoms to which they are attached form a5-6 membered heterocyclic ring having one or two ring nitrogen atoms;

each R⁹ is independently halogen, C₁-C₆-alkyl, C₃-C₆-cycloalkyl, CF₃,OCF₃, S(C₁-C₆-alkyl), CN, OCH₂-phenyl, CH₂O-phenyl, NH₂,NH—(C₁-C₆-alkyl), N—(C₁-C₆-alkyl)₂, piperidine, pyrrolidine, CH₂F, CHF₂,OCH₂F, OCHF₂, OH, SO₂(C₁-C₆-alkyl), C(O)NH₂, C(O)NH(C₁-C₆-alkyl), andC(O)N(C₁-C₆-alkyl)₂;

R¹⁰ is H or Me; and

m, n and p are independently 0 or 1.

A specific compound of Formula I is a compound wherein A is

A specific compound of Formula I is a compound Formula Ia:

or a pharmaceutically acceptable salt thereof.

In one aspect of the invention the compound of formula I excludes thecompound(S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-oneFormula Ia:

and pharmaceutically acceptable salts thereof (this compound may also bereferred to as GDC-0068).

Preparation of Formula I Compounds

Compounds of this invention may be synthesized by synthetic routes thatinclude processes analogous to those well known in the chemical arts,particularly in light of the description contained herein. The startingmaterials are generally available from commercial sources such asAldrich Chemicals (Milwaukee, Wis.) or are readily prepared usingmethods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-19, Wiley, N.Y. (1967-1999 ed.), orBeilsteins Handbuch der organischen Chemie, 4, Aufl. ed.Springer-Verlag, Berlin, including supplements).

Compounds of Formula I may be prepared singly or as compound librariescomprising at least 2, for example 5 to 1,000 compounds, or 10 to 100compounds. Libraries of compounds of Formula I may be prepared by acombinatorial ‘split and mix’ approach or by multiple parallel synthesesusing either solution phase or solid phase chemistry, by proceduresknown to those skilled in the art. Thus according to a further aspect ofthe invention there is provided a compound library comprising at least 2compounds of Formula I, or salts thereof.

For illustrative purposes, Schemes 1-4 and Schemes A-J shows a generalmethod for preparing the compounds of the present invention as well askey intermediates. For a more detailed description of the individualreaction steps, see the Examples section below. Those skilled in the artwill appreciate that other synthetic routes may be used to synthesizethe inventive compounds. Although specific starting materials andreagents are depicted in the Schemes and discussed below, other startingmaterials and reagents can be easily substituted to provide a variety ofderivatives and/or reaction conditions. In addition, many of thecompounds prepared by the methods described below can be furthermodified in light of this disclosure using conventional chemistry wellknown to those skilled in the art.

Scheme 1 shows a method of preparing compound 10 of Formula I wherein R¹is H, R² is H and R⁵ is H. Formation of pyrimidine 2 can be accomplishedby the reaction of the keto ester 1 with thiourea in the presence of abase such as KOH in an appropriate solvent, such as ethanol. Afterreduction of the mercapto group of compound 2 under standard reducingconditions (e.g., Raney Ni and NR₄OH) to provide compound 3, thehydroxypyrimidine 3 can be chlorinated under standard conditions (e.g.,POCl₃ in DIEA/DCE) to provide compound 4. Compound 4 is then oxidizedunder standard conditions (e.g., MCPBA in an appropriate solvent such asCHCl₃) to give the pyrimidine-oxide 5. Treatment of the pyrimidine-oxidewith acetic anhydride gives the rearrangement product 6. Compound 7 isobtained by reacting compound 6 with an appropriately substitutedpiperidine under standard S_(N)Ar reaction conditions to providecompound 7. Compound 7 is hydrolyzed to provide compound 8, which isthen deprotected to yield the intermediate 9. Acylation of thepiperazinyl cyclopenta[d]pyrimidine 9 with an appropriated amino acid inthe presence of a coupling reagent such as HBTU, followed bydeprotection if necessary, gives compound 10 of Formula I.

Scheme 2 shows a method of preparing compounds 22, 25 and 27 of FormulaI wherein R¹, R² and R⁵ are methyl. According to Scheme 2, brominationof (+)-pulegone 11 with bromine gives the dibromide 12. The treatment ofthe dibromide 12 with a base such as sodium ethoxide provides thepulegenate 13. Ozonolysis of the pulegenate 13 gives the ketoester 14.Treatment of the keto ester 14 with thiourea in the presence of a basesuch as KOH in ethanol, followed by reduction of the mercapto groupunder standard conditions (e.g., Raney Ni catalyst in ammonia) affordsthe hydroxypyrimidine 16. Chlorination of the hydroxypyrimidine 16 understandard conditions (e.g., POCl₃) provides the 4-chloropyrimidine 17.The oxidation of the 4-chloropyrimidine 17 with an oxidizing agent suchas MCPBA or hydrogen peroxide provides the N-oxide 18. Rearrangement ofthe N-oxide 18 with acetic anhydride yields the intermediate 19.Compound 19 is reacted with the desired piperazine according to theprocedure described in Scheme 1 to provide compound 20 where R⁵ is H and23 where R⁵ is Me. Compounds 20 and 23 are subjected to chiralseparation using HPLC with chiral stationary and then hydrolyzed upontreatment with a base such as lithium hydroxide to provide compounds 21and 24, respectively. After deprotection, compounds 21 and 24 are thenreacted with the appropriate amino acid to provide compounds 22 and 25,respectively.

Alternatively, the 7-hydroxy group of compound 24 may be alkylated withan alkylation reagent such as an alkyl halide in the presence of a basesuch as NaH or KOH to provide compound 26 where R² is Me. Afterdeprotection, compound 26 is then reacted with the appropriate aminoacid to provide compound 27.

Scheme 3 shows an alternative method of preparing compounds 73 and 74.According to Scheme 3, amination of 14 using an ammonia synthon gives63. Pyrimidine formation using, for example, ammonium formate in thepresence of formamide at 50° C.-250° C. and/or at high pressure givesthe bicyclic unit 64. Activation of 64 using, for example, POCl₃ orSOCl₂ gives the activated pyrimidine 65. Displacement of this leavinggroup, using a suitable protected/substituted piperazine at 0° C. to150° C. gives the piperazine 66. Oxidation, using, for example,m-chloroperoxybenzoic acid (“MCPBA” or “m-CPBA”) or Oxone® at −20° C. to50° C. gives the N-oxide 67. Treatment with an acylating agent (e.g.,acetic anhydride) followed by heating (40° C. to 200° C.) causesrearrangement to give 68. Hydrolysis, using, for example LiOH or NaOH at0° C. to 50° C. gives the alcohol 69. Oxidation, using for example,Swern conditions, MnO₄ or pyridine-SO₃ complex at appropriatetemperatures gives the ketone 70. Asymmetric reduction using, forexample, a catalytic chiral catalyst in the presence of hydrogen, theCBS catalyst or a borohydride reducing agent in the presence of a chiralligand gives rise to either the (R) or the (S) stereochemistry at thealcohol 71 or 72. Alternatively, a non-chiral reducing agent could beused (e.g., H₂, Pd/C), allowing the methyl group on the cyclopentaneunit to provide facial selectivity and ultimately diastereoselectivity.If the reduction gives a lower diastereoselctivity, the diastereomerscould be separated by (for example) chromatography, crystallization orderivitization. Finally deprotection of the Boc-group, using, forexample, acid at 0° C. to 50° C., acylation using an appropriatelyfunctionalized amino acid and final functionalization of the amine ofthis amino acid (e.g., removal of any protecting group, alkylation,reductive amination or acylation to introduce new substituents) givesrise to the final compounds 73 and 74.

Introduction of a chiral auxiliary (e.g., Evans oxazolidinone, etc.) tocompound 1 may be accomplished by standard acylation procedures to givethe conjugate 2. For example, treatment of the acid with an activatingagent (e.g., COCl₂) or mixed anhydride formation (e.g.,2,2-dimethylpropanoyl chloride) in the presence of an amine base at −20°C. to 100° C. followed by treatment with the appropriate chiralauxiliary (X)□ gives compound 2. The stereochemistry and choice of thechiral auxiliary may determine the stereochemistry of the newly createdchiral center and the diastereoselectivity. Treatment of compound 2 witha Lewis acid (e.g., TiCl₄) at low temperature (e.g., −20° C. to −100°C.) and an amine base (e.g., Hunig's base) followed by the use of anappropriately substituted imminium ion precursor 3 at low temperaturethen gives rise to compound 4. The temperature, Lewis acid and chiralauxiliary may all be expected to influence the diastereoselectivity ofthe addition adduct. Finally, saponification under mild conditions(e.g., LiOH/H₂O at −10° C. to 30° C.) gives rise to the desired acid 5.

According, another aspect of this invention provides a method ofpreparing a compound of Formula I, comprising:

reacting a compound having the formula:

wherein R¹, R², R⁵ and R¹⁰ are as defined herein, with an amino acidhaving the formula:

wherein R⁶, R⁷, R^(a), R^(b), R^(c), R^(d), G, m, n and p are as definedherein.

The amino acids used in the synthesis of compounds of Formula I asillustrated in Schemes 1-4 and in the Examples are either commerciallyavailable or may be prepared according to the methods disclosed herein.For example, in certain embodiments the amino acids used to preparecompounds of Formula I include β-phenylglycine amino acids having theFormula 1A, γ-phenylglycine amino acids having the Formula 2A,β-phenylalanine amino acids having the Formula 3A, and γ-phenylalanineamino acids having the Formula 4A.

Methods of preparing amino acids of Formulas 1A-4A are shown in SchemesA-J.

Scheme A illustrates a method of preparing optionally substitutedβ-phenylglycine amino acids 25 and 26 of the Formula 1A wherein R⁸ is H,and R⁶, and R⁹ and are as defined herein, t is 0 to 4, and R⁷ is H or anamine protecting group. According to Scheme A, the acid 20 is convertedto an ester 21 wherein R′ is alkyl using standard conditions such astreatment with an appropriate alcohol (e.g., MeOH) in the presence of acatalytic amount of an acid such as concentrated H₂SO₄ or a couplingagent such as DCC/DMAP; or alternatively by treatment with anappropriate electrophile (e.g., Mel, EtBr, BnBr) in the presence of abase such as NEt₃/DMAP at an appropriate temperature (e.g., −20° C. to100° C.). The appropriate choice of ester is determined by theconditions required to reform the acid at the end of the synthesis, withmany appropriate examples and conditions being listed in ‘ProtectiveGroups in Organic Synthesis’ by Greene and Wuts, Wiley-Interscience,third edition, Chapter 5. Introduction of the hydroxymethyl group toprovide compound 22 may be performed by treatment with an appropriatealdehyde (e.g., formaldehyde) in the presence of base such as NaOEt atan appropriate temperature (e.g., −20° C. to room temperature).Activation of the alcohol group of compound 22 to form a leaving group(e.g., a mesylate, tosylate, halide) may be accomplished by treatmentwith, for example, methanesulphonyl chloride in the presence of excessbase such as NEt₃, DIPEA, or DBU at an appropriate temperature (e.g.,−20° C. to room temperature). In many cases the olefin 24 can beisolated directly from this procedure, in other cases warming (30° C. to100° C.) or additional base (e.g., DBU in the case of halide) may berequired to complete the elimination to provide compound 24. Theactivated olefin 24 may be treated with the desired primary amine (e.g.,ethylamine) in a suitable solvent, such as THF, at an appropriatetemperature (e.g., −20° C. to reflux) to generate the amino esterintermediate. In the case wherein compound 24 has an electron richaromatic ring or electron poor/bulky primary amine, heating (e.g.,30-240° C. in a sealed tube) or microwave chemistry may be required.Protection of the amine group (for example as Boc-group) may beaccomplished using Boc₂O under standard conditions to provide compound23 wherein Pg is a protecting group. Alternative protecting groups maybe used, and many appropriate examples are listed in ‘Protective Groupsin Organic Synthesis’ by Greene and Wuts, Wiley-Interscience, thirdedition, Chapter 7. Saponification of the ester 23 to form the protectedamino acid 25 may be accomplished using conditions appropriate for theester (e.g., aqueous LiOH for methyl esters, hydrogenation for benzylesters, acid for t-butyl esters).

Alternatively, the activated olefin 24 may be treated with a secondaryamine (e.g., diethylamine) in a suitable solvent such as THF at anappropriate temperature (e.g., −20° C. to reflux) to generate theaminoester intermediate (not shown). In the case wherein compound 24 hasan electron rich aromatic ring or electron poor/bulky secondary amine,heating (e.g., 30-240° C. in a sealed tube) or microwave chemistry maybe required. Saponification of the ester to form the amino acid 26 maybe accomplished using conditions appropriate for the ester (e.g.,aqueous LiOH for methyl esters, hydrogenation for benzyl esters, acidfor t-butyl esters, etc.).

In an alternative to Scheme A, Pg may be substituted with R⁷ incompounds 23 and 25.

Scheme A1 shows an alternative to Scheme 1, wherein the activated olefin24 is reacted to form the amino acid 26A.

Scheme B shows a method of preparing optionally substitutedβ-phenylglycine amino acids 30 and 31 of Formula 1A wherein R⁸ is OH,and R⁶, and R⁹ are as defined herein, t is 0 to 4, and R⁷ is as definedherein or an amine protecting group. Oxidation of the unsaturated ester24 (prepared according to Scheme A), wherein t is 0-4 and R′ is alkyl,using a standard oxidizing agent such as MCPBA at an appropriatetemperature (room temperature to reflux) provides the epoxideintermediate 28. Intermediate 28 may be treated with an appropriateamine, typically at high temperature (e.g., 50-300° C.) and highpressure (e.g., in a sealed tube or a bomb) to give the amino alcohol 29or 30. If a secondary amine is used (such as in the preparation ofcompound 30), then deprotection of the ester using conditions listed in‘Protective Groups in Organic Synthesis’ by Greene and Wuts,Wiley-Interscience, third edition, Chapter 5 may be used (e.g., LiOH fora methyl ester, hydrogenation for a benzyl ester, etc). When a primaryamine is used (such as in the preparation of compound 29), protection ofthe amine (e.g., as a Boc-group using Boc anhydride) followed bydeprotection of the ester (using the above conditions) provide thehydroxylated amino acid 31.

Scheme C shows a method of preparing optionally substitutedβ-phenylglycine amino acids 36 of the Formula 1A wherein R⁸ is methyl,R⁶ is H, R⁷ is an amine protecting group t is 0 to 4, and R⁹ is asdefined herein. The ester 32, wherein R′″ is alkyl, can be treated witha base (e.g., NaOtBu) at an appropriate temperature (e.g., 0° C. toreflux) to form the anion, followed by addition of an electrophile(e.g., tert-butyl 2-bromoacetate) at an appropriate temperature (e.g.,−78° C. to room temperature) to give the homologated ester 33. Removalof the t-butyl ester of compound 33 using an appropriate acid such asTFA or HCl at an appropriate temperature (e.g, 0° C. to reflux) providescompound 34. A Curtius rearrangement of compound 34 using, for example,DPPA in the presence of mild base such as NEt₃ at an appropriatetemperature (e.g., 0° C. to reflux), followed by treatment of thereactive intermediate with an alcohol (e.g., t-BuOH), optionally in thepresence of a Lewis acid (e.g., SnCl₂) at higher temperature (e.g.,40-200° C.) provides compound 35 wherein Pg is an amine protectinggroup. The choice of alcohol used to prepare compound 35 determines theamine protecting group (e.g., t-BuOH provides the Boc-amine).Deprotection of the ester group of compound 35 using standard conditions(e.g., with LiOH when the protecting group is a methyl ester,hydrogenation for a benzyl ester, etc.) gives the acid compound 36.

In one alternative of Scheme C, R⁸ may be methyl, H or F.

In another alternative of Scheme C, Pg may be substituted with R⁷ incompounds 35 and 36.

Scheme D shows a method of preparing optionally substitutedγ-phenylglycine amino acids 40 of Formula 2A wherein R^(c), R^(d), andR⁹ are as defined herein t is 0 to 4, R⁶ is H, and R⁷ is an amineprotecting group such as Boc. The starting unsaturated ester 24,prepared according to Scheme A, can be treated with a substitutednitromethane derivative (e.g., nitroethane) in the presence of a basesuch as DBU at an appropriate temperature (e.g., 0° C. to roomtemperature) to give the homologated adduct 37. The nitro group ofcompound 37 can be reduced using standard conditions (e.g.,hydrogenation, Zn/acid, etc.) at an appropriate temperature (e.g., roomtemperature to reflux), and the resulting intermediate can be cyclizedto give the lactam intermediate 38. Protection of the amine, for examplewith a Boc-group to provide compound 39, may be accomplished using Boc₂Ounder standard conditions. Alternative protecting groups may be used,and many appropriate examples are listed in ‘Protective Groups inOrganic Synthesis’ by Greene and Wuts, Wiley-Interscience, thirdedition, Chapter 7. Treatment of compound 39 with an aqueous base suchas LiOH or KOH at an appropriate temperature (e.g., 0 to 100° C.)effects ring opening of the lactam to give the appropriately substitutedprotected amino acid compound 40.

In one alternative of Scheme D, Boc may be replaced with R⁷ in compounds39 and 40.

Scheme D1 shows representative methods of forming the singleenantionmers of the gamma amino acids 40d and 40e, wherein R^(c), R^(d),and R⁹ are as defined herein, t is 0 to 4, R⁶ is H, and R⁷ is an amineprotecting group such as Boc. In one possible method, the racemic aminoacid is subject to chiral chromatographic separation using a chiralstationary phase. Alternatively, a diastereomeric mixture may beprepared which could be separated by conventional chromatographictechniques. For example, activation of compound 40 (e.g., COCl₂, base)and introduction of a chiral auxiliary (e.g., an Evans' oxazolidinone)in the presence of a basic amine (e.g., Hunig's base) at −20° C. to 50°C. gives the diastereomeric mixture of compounds 40b and 40c. Thismixture may be separated using standard conditions (e.g., columnchromatography, HPLC, SFC, etc.) to give the individual diastereomers.These may be converted to the desired acids by cleavage of the chiralauxiliary (in the case of an Evans' auxiliary, by using (for example)LiOH/HOOH at −15° C. to room temperature) to give the compounds 40d and40e. The temperature may need to be kept low so as to preventracemisation of the newly separated chiral center.

Scheme E shows a method of making optionally substituted γ-phenylglycineamino acids 44 of Formula 2A wherein R⁸ is methyl, R⁶ is H, R⁷ is anamine protecting group, t is 0 to 4, and R⁹ is as defined herein. Theester 32, wherein R′″ is alkyl and t is 0-4, can be treated with asuitable base such as KOtBu at an appropriate temperature (e.g., 0° C.to reflux) to form the anion, followed by addition of an acrylate unit(e.g., t-butylacrylate) at a temperature ranging from −78° C. to roomtemperature to give the homologated ester 41. Saponification of thet-butyl ester of compound 41 by treatment with a suitable acid such asTFA or HCl at an appropriate temperature (e.g, 0° C. to reflux) providescompound 42. A Curtius rearrangement of compound 42 using, for example,DPPA in the presence of mild base such as NEt₃ at an appropriatetemperature (e.g., 0° C. to reflux), followed by treatment of thereactive intermediate with an appropriate alcohol (e.g., tBuOH),optionally in the presence of a Lewis acid (e.g., SnCl₂) at elevatedtemperatures (e.g., 40-200° C.) provides compound 43. The choice ofalcohol determines the amine protecting group of compound 43 (e.g.,tBuOH provides the Boc-amine). Deprotection of the ester of compound 43under standard conditions (e.g., LiOH for a methyl ester, hydrogenationfor a benzyl ester, etc.) gives the acid 44.

In one alternative to Scheme E, Pg may be substituted with R⁷ incompounds 43 and 44.

Scheme F shows a method of preparing optionally substitutedβ-phenylalanine amino acids 48, 49 and 50 of Formula 3A wherein R⁶ is H,R⁷ is an amine protecting group, t is 0 to 4, and R⁹ is as definedherein. An appropriately substituted aldehyde 45 can be treated with acyanoacetate of the formula CN—CH₂CO₂R′″ wherein R′″ is alkyl (e.g.,ethyl 2-cyanoacetate) in the presence of a suitable base such aspiperidine at an appropriate temperature (e.g., room temperature toreflux) to give the unsaturated ester 46. Reduction of the olefin andthe nitrile groups of compound 46 to provide compound 47 may beaccomplished in a number of ways. For example, the olefin may be reducedwith any agent known to effect 1,4-reductions, such as NaBH₄. Thenitrile may be reduced using agents such as LiAlH₄ or NaBH₄ in thepresence of a Lewis acid such as BF₃.OEt₂ or TFA. A number ofalternative reducing agents may be used, such as those listed in‘Reductions in Organic Chemistry’ by Hudlicky, ACS monograph, 2^(nd)edition, Chapter 18. If desired, the primary amine 47 can bemonoalkylated or bisalkylated at this stage using standard conditions(e.g., reductive amination using an appropriate aldehyde, Lewis acid andreducing agent) to provide intermediates (not shown) en route tocompounds 48 and 49. To prepare primary and secondary amines, protectionmay be accomplished using any number of protecting groups (e.g.,‘Protective Groups in Organic Synthesis’ by Greene and Wuts,Wiley-Interscience, third edition, Chapter 7), for example as aBoc-group using Boc anhydride at 0° C. to room temperature. Cleavage ofthe ester group to form the amino acid 48, 49 or 50 may be accomplishedusing an aqueous bases such as LiOH or KOH, or any of the alternativereagents listed in the aforementioned ‘Protecting Groups’ text (e.g.,hydrogenation for a benzyl ester).

In one alternative to Scheme F, Pg may be substituted with R⁷ incompounds 49 or 50.

Scheme G shows a method of preparing optionally substitutedα-phenylalanine amino acids 54 of Formula 4A, wherein R⁶ is H, R⁷ is anamine protecting group, t is 0 to 4, and R⁹ is as defined herein. Anappropriately substituted acid 51 may be reduced to the benzyl alcohol52 using for example LiAlH₄ at a temperature ranging from roomtemperature to reflux. The alcohol group of compound 52 can be activatedas a leaving group (e.g., halide, mesylate, etc.) using, for example,PBr₃, MsCl/NEt₃, etc. Displacement of this leaving group using aprotected glycine derivative such as ethyl2-(diphenylmethyleneamino)acetate in the presence of strong base such asLDA, nBuLi provides the amino ester intermediate 53 wherein R¹ is alkyland Pg is a protecting group. Appropriate protecting groups are listedin ‘Protective Groups in Organic Synthesis’ by Greene and Wuts,Wiley-Interscience). The amine protecting group may be changed at thisstage, for example to introduce a Boc-group. Subsequent deprotection ofthe ester 53 (e.g., using 3N HCl, LiOH, hydrogenation for a benzylester, etc.) at an appropriate temperature (e.g., 0° C. to reflux)provides the desired N-protected amino acid 54.

In one alternative to Scheme G, Pg may be substituted with R⁷ incompound 54 after the deprotection of compound 53.

Scheme H shows a method of preparing optionally substitutedγ-phenylglycine amino acids 56 of Formula 2A wherein R⁶ and R⁸ togetherwith the atoms to which they are attached form a spirocyclicheterocyclic ring, R⁷ is an amine protecting group, t is 0 to 4, and R⁹is as defined herein. According to Scheme H, the unsaturated ester 24can be treated with a suitably protected glycine derivative (e.g.,benzylglycine) and formaldehyde under dry conditions (e.g., withaddition of molecular sieves) at an appropriate temperature (e.g., roomtemperature to reflux) to generate compound 55. Cleavage of the benzylgroup using standard conditions (e.g., via hydrogenation,1-chloroethylformate, etc.) followed by addition of an amine protectinggroup such as a Boc-group and cleavage of the ester under standardconditions (e.g., LiOH for a methyl ester, acid for a t-butyl ester,etc., at 0° C. to reflux) provides the N-protected amino acid 56.

In one alternative to Scheme H, Pg may be substituted with R⁷ incompound 56.

Scheme I shows a method of preparing optionally substitutedβ-phenylalanine amino acids 61 and 62 of Formula 3A wherein R⁶ and R^(b)together with the atoms to which they are attached form a heterocyclicring, and R⁷ and R⁹ are as defined herein and t is 0 to 4. The acid 57is converted to an ester 58 using standard conditions such as treatmentwith an appropriate alcohol (e.g., MeOH) in the presence of eithercatalytic acid (e.g., concentrated H₂SO₄ or TMSCl) or a coupling agent(e.g., DCC/DMAP); or alternatively by treatment with an appropriateelectrophile (e.g., Mel, EtBr, BnBr) in the presence of a suitable basesuch as NEt₃/DMAP at appropriate temperatures (e.g., −20° C. to 100°C.). The appropriate choice of ester is determined by the conditionsrequired to reform the acid at the end of the synthesis, such asdescribed in ‘Protective Groups in Organic Synthesis’ by Greene andWuts, Wiley-Interscience, third edition, Chapter 5. Cyclization ofcompound 58 to provide compound 59 may be achieved using, for example,N-(methoxymethyl)(phenyl)-N-((trimethylsilyl)methyl)methanamine in thepresence of TFA. This particular set of reagents generates thebenzylamine, which can be cleaved to provide compound 60 under standardconditions such as such as hydrogenation at −20° C. to 50° C. or anyother standard conditions such as those listed in ‘Protective Groups inOrganic Synthesis’ by Greene and Wuts, Wiley-Interscience, thirdedition, Chapter 7. Protection of the free amine of compound 60 with analternative protecting group (e.g., Boc) using reagents listed in theaforementioned text, such as Boc-anhydride, followed by cleavage of theester using standard conditions appropriate for the ester (e.g., aqueousLiOH for methyl esters, hydrogenation for benzyl esters, acid fort-butyl esters) provides the acid compound 61. Alternatively, the freeamine can be functionalized further (e.g., using alkylation, reductiveamination, or acylation conditions), followed by ester cleavage togenerate the tertiary amino acid compound 62.

Either enantiomer of the b-amino acids may be prepared using a proceduresuch as that shown in Scheme J. A 2-phenylacetate coupled with anappropriate chiral auxiliary (R*) (for example, an Evans' auxiliary or aSultam) with the appropriate stereochemistry to generate the desiredchemistry at the b-position of the amino acid may be treated with animine or iminium ion synthon (e.g., prepared in situ by the presence ofa Lewis acid (e.g., TiCl₄) and an appropriately substitutedalkoxymethanamine or N-(alkoxymethyl)amide/carbamate at −100° C. to 50°C.). The asymmetric addition may require the presence of Lewis acids(e.g., TiCl₄), amine bases (e.g., Hunig's base) and lower temperatures(e.g., −100° C. to 0° C.) to generate the best levels of stereochemicalinduction. If the de is lower than required, the separate diastereomersmay be separated at this stage by (for example) chromatography orcrystallization. Cleavage of the chiral auxiliary, using methods knownto cleave the chosen auxiliary (e.g., LiOH/H₂O₂ at −50° C. to 50° C. forthe Evans auxiliary) then leads to the desired N-protected b-amino acidwith the desired stereochemistry at the b-position. Additionally, if R⁶is also a protecting group (e.g., 2,4-dimethoxybenzyl), it may beremoved in the presence of the Boc-group (e.g., hydrogenation or DDQ,etc.) to give the Boc-amino acid, which upon removal of the Boc-groupwould provide the primary amine, which may be further functionalized byalkylation, acylation or reductive amination (either prior to or aftercoupling with the pyrimidine-piperazine unit).

In preparing compounds of Formula I, protection of remotefunctionalities (e.g., primary or secondary amines, etc.) ofintermediates may be necessary. The need for such protection will varydepending on the nature of the remote functionality and the conditionsof the preparation methods. Suitable amino-protecting groups (NH-Pg)include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC),benzyloxycarbonyl (CBz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc). Theneed for such protection is readily determined by one skilled in theart. For a general description of protecting groups and their use, seeT. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons,New York, 1991.

Methods of Separation

In any of the synthetic methods for preparing compounds of Formula I, itmay be advantageous to separate reaction products from one anotherand/or from starting materials. The desired products of each step orseries of steps is separated and/or purified to the desired degree ofhomogeneity by the techniques common in the art. Typically suchseparations involve multiphase extraction, crystallization from asolvent or solvent mixture, distillation, sublimation, orchromatography. Chromatography can involve any number of methodsincluding, for example: reverse-phase and normal phase; size exclusion;ion exchange; high, medium and low pressure liquid chromatographymethods and apparatus; small scale analytical; simulated moving bed(SMB) and preparative thin or thick layer chromatography, as well astechniques of small scale thin layer and flash chromatography.

Another class of separation methods involves treatment of a reactionmixture with a reagent selected to bind to or render otherwise separablea desired product, unreacted starting material, reaction by product, orthe like. Such reagents include adsorbents or absorbents such asactivated carbon, molecular sieves, ion exchange media, or the like.Alternatively, the reagents can be acids in the case of a basicmaterial, bases in the case of an acidic material, binding reagents suchas antibodies, binding proteins, selective chelators such as crownethers, liquid/liquid ion extraction reagents (LIX), or the like.

Selection of appropriate methods of separation depends on the nature ofthe materials involved. For example, boiling point and molecular weightin distillation and sublimation, presence or absence of polar functionalgroups in chromatography, stability of materials in acidic and basicmedia in multiphase extraction, and the like. One skilled in the artwill apply techniques most likely to achieve the desired separation.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,J. Chromatogr., (1975) 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−)menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.,(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. of Chromatogr., (1990) 513:375-378). Enriched orpurified enantiomers can be distinguished by methods used to distinguishother chiral molecules with asymmetric carbon atoms, such as opticalrotation and circular dichroism.

Chemotherapeutic Agents

Certain chemotherapeutic agents have demonstrated surprising andunexpected properties in combination with a compound of formula I or apharmaceutically acceptable salt thereof in inhibiting cellularproliferation in vitro and in vivo. Such chemotherapeutic agentsinclude: 5-FU, a platinum agent, irinotecan, docetaxel, doxorubicin,gemcitabine, SN-38, capecitabine, temozolomide, erlotinib, PD-0325901,paclitaxel, bevacizumab, pertuzumab, tamoxifen, rapamycin, lapatinib,PLX-4032, MDV3100, abiraterone, and GDC-0973.

5-FU (fluorouracil, 5-fluorouracil, CAS Reg. No. 51-21-8) is athymidylate synthase inhibitor and has been used for decades in thetreatment of cancer, including colorectal and pancreatic cancer (U.S.Pat. No. 2,802,005; U.S. Pat. No. 2,885,396; Duschinsky et al (1957) J.Am. chem. Soc. 79:4559; Hansen, R. M. (1991) Cancer Invest. 9:637-642).5-FU is named as 5-fluoro-1H-pyrimidine-2,4-dione.

Carboplatin (CAS Reg. No. 41575-94-4) is a chemotherapeutic drug usedagainst ovarian carcinoma, lung, head and neck cancers (U.S. Pat. No.4,140,707; Calvert et al (1982) Cancer Chemother. Pharmacol. 9:140;Harland et al (1984) Cancer Res. 44:1693). Carboplatin is named asazanide; cyclobutane-1,1-dicarboxylic acid; platinum.

Cisplatin, cisplatinum, or cis-diamminedichloroplatinum(II) (CAS Reg.No. 15663-27-1) is a chemotherapeutic drug used to treat various typesof cancers, including sarcomas, some carcinomas (e.g., small cell lungcancer, and ovarian cancer), lymphomas, and germ cell tumors. It was thefirst member of a class of platinum-containing anti-cancer drugs, whichnow also includes carboplatin and oxaliplatin. Cisplatin has thestructure cis-PtCl₂(NH₃)₂.

Oxaliplatin (CAS Reg. No. 63121-00-6) is a coordination complex that isused in cancer chemotherapy (U.S. Pat. No. 4,169,846). Oxaliplatin hasbeen compared with other platinum compounds (Cisplatin, Carboplatin) inadvanced cancers (gastric, ovarian). Oxaliplatin is typicallyadministered with fluorouracil and leucovorin in a combination known asFOLFOX for the treatment of colorectal cancer.

Irinotecan (CAS Reg. No. 97682-44-5) is a topoisomerase 1 inhibitor,which prevents DNA from unwinding. Irinotecan is activated by hydrolysisto SN-38, an inhibitor of topoisomerase I. The inhibition oftopoisomerase I by the active metabolite SN-38 eventually leads toinhibition of both DNA replication and transcription. Its main use is incolon cancer, in particular, in combination with other chemotherapyagents. This includes the regimen FOLFIRI, which consists of infusional5-fluorouracil, leucovorin, and irinotecan.

Doxorubicin (CAS Reg. No. 23214-92-8) is an anthracycline antibiotic.Like all anthracyclines, it works by intercalating DNA. Doxorubicin iscommonly used in the treatment of a wide range of cancers, includinghematological malignancies, many types of carcinoma, and soft tissuesarcomas. Doxorubicin is named as(8S,10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione.

Docetaxel (CAS Reg. No. 114977-28-5) is used to treat breast, ovarian,and NSCLC cancers (U.S. Pat. No. 4,814,470; U.S. Pat. No. 5,438,072;U.S. Pat. No. 5,698,582; U.S. Pat. No. 5,714,512; U.S. Pat. No.5,750,561; Mangatal et al (1989) Tetrahedron 45:4177; Ringel et al(1991) J. Natl. Cancer Inst. 83:288; Bissery et al (1991) Cancer Res.51:4845; Herbst et al (2003) Cancer Treat. Rev. 29:407-415; Davies et al(2003) Expert. Opin. Pharmacother. 4:553-565). Docetaxel is named as(2R,3S)—N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with5, 20-epoxy-1, 2, 4, 7, 10, 13-hexahydroxytax-11-en-9-one 4-acetate2-benzoate, trihydrate (U.S. Pat. No. 4,814,470; EP 253738; CAS Reg. No.114977-28-5).

Gemcitabine (CAS Reg. No. 95058-81-4) is a nucleoside analog whichblocks DNA replication, is used to treat various carcinomas includingpancreatic, breast, NSCLC, and lymphomas (U.S. Pat. No. 4,808,614; U.S.Pat. No. 5,464,826; Hertel et al (1988) J. Org. Chem. 53:2406; Hertel etal (1990) Cancer Res. 50:4417; Lund et al (1993) Cancer Treat. Rev.19:45-55). Gemcitabine is named as4-amino-1-[3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-1H-pyrimidin-2-one.

SN-38 (CAS Reg. No. 86639-52-3) is the active metabolite of irinotecan(see above). It is 200 times more active than irinotecan itself. It hasthe name 7-ethyl-10-hydroxy-camptothecin.

Capecitabine (CAS Reg. No. 154361-50-9) is an orally-administeredchemotherapeutic agent used in the treatment of metastatic breast andcolorectal cancers. Capecitabine is a prodrug, that is enzymaticallyconverted to 5-fluorouracil in the tumor, where it inhibits DNAsynthesis and slows growth of tumor tissue. The activation ofcapecitabine follows a pathway with three enzymatic steps and twointermediary metabolites, 5′-deoxy-5-fluorocytidine (5′-DFCR) and5′-deoxy-5-fluorouridine (5′-DFUR), to form 5-fluorouracil. Capecitabinehas the namepentyl[1-(3,4-dihydroxy-5-methyl-tetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]aminomethanoate.

Temozolomide (CAS Reg. No. 85622-93-1) is an alkylating agent which canbe used for the treatment of Grade IV astrocytoma, also known asglioblastoma multiforme as well as Melanoma, a form of skin cancer.Temozolomide has the name 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide.

Erlotinib (CAS Reg. No. 183321-74-6, TARCEVA®, OSI-774, Genentech, Inc.)is used to treat non-small cell lung cancer (NSCLC), lung cancer,pancreatic cancer and several other types of cancer by specificallytargeting the epidermal growth factor receptor (EGFR) tyrosine kinase(U.S. Pat. No. 5,747,498; U.S. Pat. No. 6,900,221; Moyer et al (1997)Cancer Res. 57:4838; Pollack et al (1999) J. Pharmcol. Exp. Ther.291:739; Perez-Soler et al (2004) J. Clin. Oncol. 22:3238; Kim et al(2002) Curr. Opin. Invest. Drugs 3:1385-1395; Blackhall et al (2005)Expert Opin. Pharmacother. 6:995-1002). Erlotinib is named asN-(3-ethynylphenyl)-6,7-bis(methoxymethoxy)quinazolin-4-amine (CAS Reg.No. 183321-74-6) and has the structure:

PD-0325901 (CAS Reg. No. 391210-10-9, Pfizer) is a second-generation,non-ATP competitive, allosteric MEK inhibitor for the potential oraltablet treatment of cancer (U.S. Pat. No. 6,960,614; U.S. Pat. No.6,972,298; US 2004/147478; US 2005/085550). Phase II clinical trialshave been conducted for the potential treatment of breast tumors, colontumors, and melanoma. PD-0325901 is named as(R)—N-(2,3-dihydroxypropoxy)-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)benzamide,and has the structure:

Paclitaxel (CAS Reg. No. 33069-62-4, TAXOL®, Bristol-Myers SquibbOncology, Princeton N.J.) is isolated the compound from the bark of thePacific yew tree, Taxus brevifolia, and used to treat lung, ovarian,breast cancer, and advanced forms of Kaposi's sarcoma (Wani et al (1971)J. Am. Chem. Soc. 93:2325; Mekhail et al (2002) Expert. Opin.Pharmacother. 3:755-766). Paclitaxel is named asβ-(benzoylamino)-α-hydroxy-,6,12b-bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclodeca(3,4)benz(1,2-b)oxet-9-ylester,(2aR-(2a-α,4-β,4a-β,6-β,9-α(α-R*,β-S*),11-α,12-α,12a-α,2b-α))benzenepropanoic acid, and has thestructure:

Bevacizumab (CAS Reg. No. 216974-75-3, AVASTIN®, Genentech) is arecombinant humanized monoclonal antibody against VEGF, vascularendothelial growth factor (U.S. Pat. No. 6,054,297; Presta et al (1997)Cancer Res. 57:4593-4599). It is used in the treatment of cancer, whereit inhibits tumor growth by blocking the formation of new blood vessels.Bevacizumab was the first clinically available angiogenesis inhibitor inthe United States, approved by the FDA in 2004 for use in combinationwith standard chemotherapy in the treatment of metastatic colon cancerand most forms of metastatic non-small cell lung cancer. Severallate-stage clinical studies are underway to determine its safety andeffectiveness for patients with: adjuvant/non-metastatic colon cancer,metastatic breast cancer, metastatic renal cell carcinoma, metastaticglioblastoma multiforme, metastatic ovarian cancer, metastatichormone-refractory prostate cancer, and metastatic or unresectablelocally advanced pancreatic cancer (Ferrara et al (2004) Nat. Rev. DrugDisc. 3:391-400). Bevacizumab includes mutated human IgG1 frameworkregions and antigen-binding complementarity-determining regions from themurine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding ofhuman VEGF to its receptors. Bevacizumab has a molecular mass of about149,000 daltons and is glycosylated.

Bevacizumab and other humanized anti-VEGF antibodies are furtherdescribed in U.S. Pat. No. 6,884,879. Additional anti-VEGF antibodiesinclude the G6 or B20 series antibodies, e.g., G6-31, B20-4.1, (WO2005/012359; WO 2005/044853; U.S. Pat. No. 7,060,269; U.S. Pat. No.6,582,959; U.S. Pat. No. 6,703,020; U.S. Pat. No. 6,054,297; WO98/45332; WO 96/30046; WO 94/10202; EP 0666868B1; US 2006/009360; US2005/0186208; US 2003/0206899; US 2003/0190317; US 2003/0203409;20050112126; Popkov et al (2004) Journal of Immunological Methods288:149-164. A “B20 series antibody” is an anti-VEGF antibody that isderived from a sequence of the B20 antibody or a B20-derived antibodyaccording to any one of FIGS. 27-29 of WO 2005/012359, the entiredisclosure of which is expressly incorporated herein by reference. Inone embodiment, the B20 series antibody binds to a functional epitope onhuman VEGF comprising residues F17, M18, D19, Y21, Y25, Q89, I91, K101,E103, and C104. Other anti-VEGF antibodies include those that bind to afunctional epitope on human VEGF comprising residues F17, M18, D19, Y21,Y25, Q89, I91, K101, E103, and C104 or, alternatively, comprisingresidues F17, Y21, Q22, Y25, D63, 183 and Q89.

Trastuzumab (HERCEPTIN®, huMAb4D5-8, rhuMAb HER2, Genentech) is arecombinant DNA-derived humanized, IgG1 kappa, monoclonal antibodyversion of the murine HER2 antibody which selectively binds with highaffinity in a cell-based assay (Kd=5 nM) to the extracellular domain ofthe human epidermal growth factor receptor2 protein, HER2 (ErbB2) (U.S.Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213;U.S. Pat. No. 6,639,055; Coussens L, et al (1985) Science 230:1132-9;Slamon D J, et al (1989) Science 244:707-12). Trastuzumab contains humanframework regions with the complementarity-determining regions of amurine antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2antigen and thus inhibits the growth of cancerous cells. Trastuzumab hasbeen shown, in both in vitro assays and in animals, to inhibit theproliferation of human tumor cells that overexpress HER2 (Hudziak R M,et al (1989) Mol Cell Biol 9:1165-72; Lewis G D, et al (1993) CancerImmunol Immunother; 37:255-63; Baselga J, et al (1998) Cancer Res.58:2825-2831). Trastuzumab is a mediator of antibody-dependent cellularcytotoxicity, ADCC (Hotaling T E, et al (1996) [abstract]. Proc. AnnualMeeting Am Assoc Cancer Res; 37:471; Pegram M D, et al (1997)[abstract]. Proc Am Assoc Cancer Res; 38:602; Sliwkowski et al (1999)Seminars in Oncology 26(4), Suppl 12:60-70; Yarden Y. and Sliwkowski, M.(2001) Nature Reviews: Molecular Cell Biology, Macmillan Magazines,Ltd., Vol. 2:127-137). HERCEPTIN® was approved in 1998 for the treatmentof patients with ErbB2-overexpressing metastatic breast cancers (Baselgaet al, (1996) J. Clin. Oncol. 14:737-744). The FDA approved HERCEPTIN®in 2006 as part of a treatment regimen containing doxorubicin,cyclophosphamide and paclitaxel for the adjuvant treatment of patientswith HER2-positive, node-positive breast cancer. There is a significantclinical need for developing further HER2-directed cancer therapies forthose patients with HER2-overexpressing tumors or other diseasesassociated with HER2 expression that do not respond, or respond poorly,to HERCEPTIN® treatment.

Pertuzumab (OMNITARG™, rhuMab 2C4, Genentech) is a clinical stage,humanized antibody and the first in a new class of agents known as HERdimerization inhibitors (HDIs) which block the ability of the HER2receptor to collaborate with other HER receptor family members, i.e.HER1/EGFR, HER3, and HER4 (U.S. Pat. No. 6,949,245; Agus et al (2002)Cancer Cell 2:127-37; Jackson et al (2004) Cancer Res 64:2601-9; Takaiet al (2005) Cancer 104:2701-8). In cancer cells, interfering withHER2's ability to collaborate with other HER family receptors blockscell signaling and may ultimately lead to cancer cell growth inhibitionand death of the cancer cell. HDIs, because of their unique mode ofaction, have the potential to work in a wide variety of tumors,including those that do not overexpress HER2 (Mullen et al (2007)Molecular Cancer Therapeutics 6:93-100).

Temozolomide, (CAS Reg. No. 85622-93-1, TEMODAR®, TEMODAL®, ScheringPlough) is a oral chemotherapy drug approved by the FDA for thetreatment of anaplastic astrocytoma, and has been studied for otherbrain tumor types such as glioblastoma multiforme (U.S. Pat. No.5,260,291; Stevens et al (1984) J. Med. Chem. 27:196; Newlands et al(1997) Cancer Treat. Rev. 23:35-61; Danson et al (2001) Expert Rev.Anticancer Ther. 1:13-19). Temozolomide is named as(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide or3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide(U.S. Pat. No. 5,260,291, CAS No. 85622-93-1), and has the structure:

Tamoxifen (CAS Reg. No. 10540-29-1, NOLVADEX®, ISTUBAL®, VALODEX®) is anorally active, selective estrogen receptor modulator (SERM) which isused in the treatment of breast cancer and is currently the world'slargest selling drug for this indication. Tamoxifen (Nolvadex®) wasfirst approved by the FDA (ICI Pharmaceuticals, now AstraZeneca) in 1977for treatment of metastatic breast cancer (Jordan V C (2006) Br JPharmacol 147 (Suppl 1): S269-76). Tamoxifen is currently used for thetreatment of both early and advanced estrogen receptor (ER) positivebreast cancer in pre- and post-menopausal women (Jordan V C (1993) Br JPharmacol 110 (2): 507-17). It is also approved by the FDA for theprevention of breast cancer in women at high risk of developing thedisease and for the reduction of contralateral (in the opposite breast)breast cancer. Tamoxifen is named as(Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine, (CASReg. No. 10540-29-1) and has the structure:

Rapamycin (CAS Reg. No. 53123-88-9, sirolimus, RAPAMUNE®) is animmunosuppressant drug used to prevent rejection in organtransplantation, and is especially useful in kidney transplants.Rapamycin is a macrolide antibiotic (“-mycin”) first discovered as aproduct of the bacterium Streptomyces hygroscopicus in a soil samplefrom an island called Rapa Nui, better known as Easter Island (PritchardD I (2005). Drug Discovery Today 10 (10): 688-691). Rapamycin inhibitsthe response to interleukin-2 (IL-2) and thereby blocks activation of T-and B-cells. The mode of action of rapamycin is to bind the cytosolicprotein FK-binding protein 12 (FKBP12). The rapamycin-FKBP12 complexinhibits the mammalian target of rapamycin (mTOR) pathway throughdirectly binding the mTOR Complex1 (mTORC1). mTOR is also called FRAP(FKBP-rapamycin associated protein) or RAFT (rapamycin and FKBP target).Rapamycin is named as(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone(CAS Reg. No. 53123-88-9), and has the structure:

Lapatinib (CAS Reg. No. 388082-78-8, TYKERB®, GW572016, GlaxoSmithKline) has been approved for use in combination with capecitabine(XELODA®, Roche) for the treatment of patients with advanced ormetastatic breast cancer whose tumors over-express HER2 (ErbB2) and whohave received prior therapy including an anthracycline, a taxane andtrastuzumab. Lapatinib is an ATP-competitive epidermal growth factor(EGFR) and HER2/neu (ErbB-2) dual tyrosine kinase inhibitor (U.S. Pat.No. 6,727,256; U.S. Pat. No. 6,713,485; U.S. Pat. No. 7,109,333; U.S.Pat. No. 6,933,299; U.S. Pat. No. 7,084,147; U.S. Pat. No. 7,157,466;U.S. Pat. No. 7,141,576) which inhibits receptor autophosphorylation andactivation by binding to the ATP-binding pocket of the EGFR/HER2 proteinkinase domain. Lapatinib is named asN-(3-chloro-4-(3-fluorobenzyloxy)phenyl)-6-(5-((2-(methylsulfonyl)ethylamino)methyl)furan-2-yl)quinazolin-4-amine,and has the structure:

PLX-4032 (CAS Reg. No. 1029872-55-5) has been shown to cause programmedcell death in melanoma cell lines. PLX-4032 interrupts the B-Raf/MEKstep on the B-Raf/MEK/ERK pathway—if the B-Raf has the common V600Emutation. PLX-4032 works in melanoma patients whose cancer has a V600EBRAF mutation (that is, at amino acid position number 600 on the B-RAFprotein, the normal valine is replaced by glutamic acid). About 60% ofmelanomas have the V600E BRAF mutation. PLX-4032 has the followingstructure:

MDV3100 (CAS Reg. No. 915087-33-1) is an androgen receptor antagonistdrug developed for the treatment of hormone-refractory prostate cancer.Up to an 89% decrease in prostate specific antigen serum levels has beenreported after a month of taking the medicine. As opposed tobicalutamide, MDV3100 does not promote translocation of AR to thenucleus and in addition prevents binding of AR to DNA and AR tocoactivator proteins. MDV 3100 was found clinically active formetastatic castration-resistant prostate cancer patients in ongoingphase I and II trials. MDV3100 has the name4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-methylbenzamide.

Abiraterone (CAS Reg. No. 154229-19-3; see U.S. Pat. Nos. 5,604,213 and5,618,807) is a drug currently under investigation for use incastration-resistant prostate cancer. It blocks the formation oftestosterone by inhibiting CYP17A1 (CYP450c17), an enzyme also known as17α-hydroxylase/17,20 lyase. This enzyme is involved in the formation ofDHEA and androstenedione, which may ultimately be metabolized intotestosterone. Abiraterone has the name(3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17-(pyridin-3-yl)-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[α]phenanthren-3-ol.It may also be administered as the acetate prodrug(3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17-(pyridin-3-yl)-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthren-3-ylacetate.

GDC-0973 is a selective inhibitor of MEK, also known as mitogenactivated protein kinase kinase (MAPKK), which is a key component of theRAS/RAF/MEK/ERK pathway that is frequently activated in human tumors.Inappropriate activation of the MEK/ERK pathway promotes cell growth inthe absence of exogenous growth factors. A Phase I clinical trialevaluating GDC-0973 for solid tumors is ongoing. GDC-0973 can beprepared as described in International Patent Application PublicationNumber WO2007044515(A1). GDC-0973 has the name:(S)-(3,4-difluoro-2-(2-fluoro-4-iodophenylamino)phenyl)(3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl)methanone,and the following structure:

Pharmaceutical Compositions

Pharmaceutical compositions or formulations of the present inventioninclude combinations of Formula I compounds, a chemotherapeutic agent,and one or more pharmaceutically acceptable carrier, glidant, diluent,or excipient.

The Formula I compounds, and chemotherapeutic agents of the presentinvention may exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike, and it is intended that the invention embrace both solvated andunsolvated forms.

The Formula I compounds, and chemotherapeutic agents of the presentinvention may also exist in different tautomeric forms, and all suchforms are embraced within the scope of the invention. The term“tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Pharmaceutical compositions encompass both the bulk composition andindividual dosage units comprised of more than one (e.g., two)pharmaceutically active agents including a Formula I compound and achemotherapeutic agent selected from the lists of the additional agentsdescribed herein, along with any pharmaceutically inactive excipients,diluents, carriers, or glidants. The bulk composition and eachindividual dosage unit can contain fixed amounts of the aforesaidpharmaceutically active agents. The bulk composition is material thathas not yet been formed into individual dosage units. An illustrativedosage unit is an oral dosage unit such as tablets, pills, capsules, andthe like. Similarly, the herein-described method of treating a patientby administering a pharmaceutical composition of the present inventionis also intended to encompass the administration of the bulk compositionand individual dosage units.

Pharmaceutical compositions also embrace isotopically-labeled compoundsof the present invention which are identical to those recited herein,but for the fact that one or more atoms are replaced by an atom havingan atomic mass or mass number different from the atomic mass or massnumber usually found in nature. All isotopes of any particular atom orelement as specified are contemplated within the scope of the compoundsof the invention, and their uses. Exemplary isotopes that can beincorporated into compounds include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine,such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, ³³P, ³⁵S,¹⁸F, ³⁶Cl, ¹²³I and ¹²⁵I. Certain isotopically-labeled compounds of thepresent invention (e.g., those labeled with ³H and ¹⁴C) are useful incompound and/or substrate tissue distribution assays. Tritiated (³H) andcarbon-14 (¹⁴C) isotopes are useful for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (²H) may afford certain therapeutic advantages resulting fromgreater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸Fare useful for positron emission tomography (PET) studies to examinesubstrate receptor occupancy. Isotopically labeled compounds of thepresent invention can generally be prepared by following proceduresanalogous to those disclosed in the Schemes and/or in the Examplesherein below, by substituting an isotopically labeled reagent for anon-isotopically labeled reagent.

Formula I compounds and chemotherapeutic agents are formulated inaccordance with standard pharmaceutical practice for use in atherapeutic combination for therapeutic treatment (includingprophylactic treatment) of hyperproliferative disorders in mammalsincluding humans. The invention provides a pharmaceutical compositioncomprising a Formula I compound in association with one or morepharmaceutically acceptable carrier, glidant, diluent, or excipient.

Suitable carriers, diluents and excipients are well known to thoseskilled in the art and include materials such as carbohydrates, waxes,water soluble and/or swellable polymers, hydrophilic or hydrophobicmaterials, gelatin, oils, solvents, water and the like. The particularcarrier, diluent or excipient used will depend upon the means andpurpose for which the compound of the present invention is beingapplied. Solvents are generally selected based on solvents recognized bypersons skilled in the art as safe (GRAS) to be administered to amammal. In general, safe solvents are non-toxic aqueous solvents such aswater and other non-toxic solvents that are soluble or miscible inwater. Suitable aqueous solvents include water, ethanol, propyleneglycol, polyethylene glycols (e.g., PEG 400, PEG 300), etc. and mixturesthereof. The formulations may also include one or more buffers,stabilizing agents, surfactants, wetting agents, lubricating agents,emulsifiers, suspending agents, preservatives, antioxidants, opaquingagents, glidants, processing aids, colorants, sweeteners, perfumingagents, flavoring agents and other known additives to provide an elegantpresentation of the drug (i.e., a compound of the present invention orpharmaceutical composition thereof) or aid in the manufacturing of thepharmaceutical product (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a Formula I compound having the desired degree of purity mayoptionally be mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences (1995)18th edition, Mack Publ. Co., Easton, Pa.), in the form of a lyophilizedformulation, milled powder, or an aqueous solution. Formulation may beconducted by mixing at ambient temperature at the appropriate pH, and atthe desired degree of purity, with physiologically acceptable carriers,i.e., carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8.

The pharmaceutical formulation is preferably sterile. In particular,formulations to be used for in vivo administration must be sterile. Suchsterilization is readily accomplished by filtration through sterilefiltration membranes.

The pharmaceutical formulation ordinarily can be stored as a solidcomposition, a lyophilized formulation or as an aqueous solution.

The pharmaceutical formulations will be dosed and administered in afashion, i.e., amounts, concentrations, schedules, course, vehicles androute of administration, consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the compound to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat the coagulation factormediated disorder. Such amount is preferably below the amount that istoxic to the host or renders the host significantly more susceptible tobleeding.

As a general proposition, the initial pharmaceutically effective amountof the Formula I compound administered orally or parenterally per dosewill be in the range of about 0.01-1000 mg/kg, namely about 0.1 to 20mg/kg of patient body weight per day, with the typical initial range ofcompound used being 0.3 to 15 mg/kg/day. The dose of the Formula Icompound and the dose of the chemotherapeutic agent to be administeredmay range for each from about 1 mg to about 1000 mg per unit dosageform, or from about 10 mg to about 100 mg per unit dosage form. Thedoses of Formula I compound and the chemotherapeutic agent mayadministered in a ratio of about 1:50 to about 50:1 by weight, or in aratio of about 1:10 to about 10:1 by weight.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 18th edition, (1995) Mack Publ. Co.,Easton, Pa.

Sustained-release preparations of Formula I compounds may be prepared.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D (−) 3-hydroxybutyric acid.

The pharmaceutical formulations include those suitable for theadministration routes detailed herein. The formulations may convenientlybe presented in unit dosage form and may be prepared by any of themethods well known in the art of pharmacy. Techniques and formulationsgenerally are found in Remington's Pharmaceutical Sciences 18^(th) Ed.(1995) Mack Publishing Co., Easton, Pa. Such methods include the step ofbringing into association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations of a compound of Formula I and/or chemotherapeutic agentsuitable for oral administration may be prepared as discrete units suchas pills, hard or soft e.g., gelatin capsules, cachets, troches,lozenges, aqueous or oil suspensions, dispersible powders or granules,emulsions, syrups or elixirs each containing a predetermined amount of acompound of Formula I and/or a chemotherapeutic agent. The amount ofcompound of Formula I and the amount of chemotherapeutic agent may beformulated in a pill, capsule, solution or suspension as a combinedformulation. Alternatively, the Formula I compound and thechemotherapeutic agent may be formulated separately in a pill, capsule,solution or suspension for administration by alternation.

Formulations may be prepared according to any method known to the artfor the manufacture of pharmaceutical compositions and such compositionsmay contain one or more agents including sweetening agents, flavoringagents, coloring agents and preserving agents, in order to provide apalatable preparation. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom.

Tablet excipients of a pharmaceutical formulation may include: Filler(or diluent) to increase the bulk volume of the powdered drug making upthe tablet; Disintegrants to encourage the tablet to break down intosmall fragments, ideally individual drug particles, when it is ingestedand promote the rapid dissolution and absorption of drug; Binder toensure that granules and tablets can be formed with the requiredmechanical strength and hold a tablet together after it has beencompressed, preventing it from breaking down into its component powdersduring packaging, shipping and routine handling; Glidant to improve theflowability of the powder making up the tablet during production;Lubricant to ensure that the tableting powder does not adhere to theequipment used to press the tablet during manufacture. They improve theflow of the powder mixes through the presses and minimize friction andbreakage as the finished tablets are ejected from the equipment;Antiadherent with function similar to that of the glidant, reducingadhesion between the powder making up the tablet and the machine that isused to punch out the shape of the tablet during manufacture; Flavorincorporated into tablets to give them a more pleasant taste or to maskan unpleasant one, and Colorant to aid identification and patientcompliance.

Tablets containing the active ingredient in admixture with non-toxicpharmaceutically acceptable excipient which are suitable for manufactureof tablets are acceptable. These excipients may be, for example, inertdiluents, such as calcium or sodium carbonate, lactose, calcium orsodium phosphate; granulating and disintegrating agents, such as maizestarch, or alginic acid; binding agents, such as starch, gelatin oracacia; and lubricating agents, such as magnesium stearate, stearic acidor talc. Tablets may be uncoated or may be coated by known techniquesincluding microencapsulation to delay disintegration and adsorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base.

If desired, the aqueous phase of the cream base may include a polyhydricalcohol, i.e., an alcohol having two or more hydroxyl groups such aspropylene glycol, butane 1,3-diol, mannitol, sorbitol, glycerol andpolyethylene glycol (including PEG 400) and mixtures thereof. Thetopical formulations may desirably include a compound which enhancesabsorption or penetration of the active ingredient through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethyl sulfoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner, including a mixture of atleast one emulsifier with a fat or an oil, or with both a fat and anoil. Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. Together, theemulsifier(s) with or without stabilizer(s) make up an emulsifying wax,and the wax together with the oil and fat comprise an emulsifyingointment base which forms the oily dispersed phase of creamformulations. Emulsifiers and emulsion stabilizers suitable for use inthe formulation include Tween® 60, Span® 80, cetostearyl alcohol, benzylalcohol, myristyl alcohol, glyceryl monostearate and sodium laurylsulfate.

Aqueous suspensions of the pharmaceutical formulations contain theactive materials in admixture with excipients suitable for themanufacture of aqueous suspensions. Such excipients include a suspendingagent, such as sodium carboxymethylcellulose, croscarmellose, povidone,methylcellulose, hydroxypropyl methylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Pharmaceutical compositions may be in the form of a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension may be formulated according to the known artusing those suitable dispersing or wetting agents and suspending agentswhich have been mentioned above. The sterile injectable preparation maybe a solution or a suspension in a non-toxic parenterally acceptablediluent or solvent, such as a solution in 1,3-butanediol or preparedfrom a lyophilized powder. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile fixed oils may conventionally beemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid may likewise be used in thepreparation of injectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

The compound of formula I or a pharmaceutically acceptable salt thereofmay be employed in combination with other chemotherapeutic agents forthe treatment of a hyperproliferative disease or disorder, includingtumors, cancers, and neoplastic tissue, along with pre-malignant andnon-neoplastic or non-malignant hyperproliferative disorders. In certainembodiments, a compound of Formula I or a pharmaceutically acceptablesalt thereof is combined in a dosing regimen as combination therapy,with a second compound that has anti-hyperproliferative properties orthat is useful for treating the hyperproliferative disorder. The secondcompound of the dosing regimen preferably has complementary activitiesto the compound of formula I or a pharmaceutically acceptable saltthereof, and such that they do not adversely affect each other. Suchcompounds may be administered in amounts that are effective for thepurpose intended. In one embodiment, the therapeutic combination isadministered by a dosing regimen wherein the therapeutically effectiveamount of a compound of formula I, or a pharmaceutically acceptable saltthereof is administered in a range from twice daily to once every threeweeks (q3 wk), and the therapeutically effective amount of thechemotherapeutic agent is administered in a range from twice daily toonce every three weeks.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulation,and consecutive administration in either order, wherein preferably thereis a time period while both (or all) active agents simultaneously exerttheir biological activities.

In one specific aspect of the invention, the compound of formula I orthe pharmaceutically acceptable salt thereof can be administered for atime period of about 1 to about 10 days after administration of the oneor more agents begins. In another specific aspect of the invention, thecompound of formula I or the pharmaceutically acceptable salt thereofcan be administered for a time period of about 1 to 10 days beforeadministration of the combination begins. In another specific aspect ofthe invention, administration of the compound of formula I or thepharmaceutically acceptable salt thereof and administration of thechemotherapeutic agent begin on the same day.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments, such as to increase the therapeutic index or mitigatetoxicity or other side-effects or consequences.

In a particular embodiment of anti-cancer therapy, a compound of formulaI, or pharmaceutically acceptable salt thereof, may be combined with achemotherapeutic agent, as well as combined with surgical therapy andradiotherapy. The amounts of the compound of formula I or apharmaceutically acceptable salt thereof and the other pharmaceuticallyactive chemotherapeutic agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect.

Administration of Pharmaceutical Compositions

The compounds may be administered by any route appropriate to thecondition to be treated. Suitable routes include oral, parenteral(including subcutaneous, intramuscular, intravenous, intraarterial,inhalation, intradermal, intrathecal, epidural, and infusiontechniques), transdermal, rectal, nasal, topical (including buccal andsublingual), vaginal, intraperitoneal, intrapulmonary and intranasal.Topical administration can also involve the use of transdermaladministration such as transdermal patches or iontophoresis devices.

Formulation of drugs is discussed in Remington's PharmaceuticalSciences, 18^(th) Ed., (1995) Mack Publishing Co., Easton, Pa. Otherexamples of drug formulations can be found in Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol 3,2^(nd) Ed., New York, N.Y. For local immunosuppressive treatment, thecompounds may be administered by intralesional administration, includingperfusing or otherwise contacting the graft with the inhibitor beforetransplantation. It will be appreciated that the preferred route mayvary with for example the condition of the recipient. Where the compoundis administered orally, it may be formulated as a pill, capsule, tablet,etc. with a pharmaceutically acceptable carrier, glidant, or excipient.Where the compound is administered parenterally, it may be formulatedwith a pharmaceutically acceptable parenteral vehicle or diluent, and ina unit dosage injectable form, as detailed below.

A dose to treat human patients may range from about 20 mg to about 1600mg per day of the compound of formula I or a pharmaceutically acceptablesalt thereof. A typical dose may be about 50 mg to about 800 mg of thecompound. A dose may be administered once a day (QD), twice per day(BID), or more frequently, depending on the pharmacokinetic (PK) andpharmacodynamic (PD) properties, including absorption, distribution,metabolism, and excretion of the particular compound. In addition,toxicity factors may influence the dosage and administration dosingregimen. When administered orally, the pill, capsule, or tablet may beingested twice daily, daily or less frequently such as weekly or onceevery two or three weeks for a specified period of time. The regimen maybe repeated for a number of cycles of therapy.

Methods of Treatment

Therapeutic combinations of: (1) a compound of formula I or apharmaceutically acceptable salt thereof, and (2) a chemotherapeuticagent are useful for treating diseases, conditions and/or disordersincluding, but not limited to, those modulated by AKT kinase in amammal. Cancers which can be treated according to the methods of thisinvention include, but are not limited to, mesothelioma, endometrial,breast, lung, ovarian, prostate (including castration resistant prostacecancer “CRPC”), pancreatic, melanoma, gastric, colon, glioma, head andneck. Therapeutic combinations of: (1) a compound of formula I or apharmaceutically acceptable salt thereof, and (2) erlotinib or apharmaceutically acceptable salt thereof are useful for treatingdiseases, conditions and/or disorders including, but not limited to,those modulated by AKT kinase, and optionally also modulated by mutatedEGFR kinase, in a mammal. Cancers which can be treated according to themethods of this invention include, but are not limited to, mesothelioma,endometrial, breast, lung, ovarian, prostate (including castrationresistant prostace cancer “CRPC”), pancreatic, melanoma, gastric, colon,glioma, head and neck

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing a compound of formula I or pharmaceutically acceptablesalt thereof useful for the treatment of the diseases and disordersdescribed above is provided. In one embodiment, the kit comprises acontainer and a compound of formula I or pharmaceutically acceptablesalt thereof.

The kit may further comprise a label or package insert, on or associatedwith the container. The term “package insert” is used to refer toinstructions customarily included in commercial packages of therapeuticproducts, that contain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch therapeutic products. Suitable containers include, for example,bottles, vials, syringes, blister pack, etc. The container may be formedfrom a variety of materials such as glass or plastic. The container mayhold a compound of formula I or pharmaceutically acceptable saltthereof, or a formulation thereof which is effective for treating thecondition and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a compound of formula I or a pharmaceutically acceptablesalt thereof. The label or package insert indicates that the compositionis used for treating the condition of choice, such as cancer. In oneembodiment, the label or package inserts indicates that the compositioncomprising a compound of formula I or pharmaceutically acceptable saltthereof can be used to treat a disorder resulting from abnormal cellgrowth. The label or package insert may also indicate that thecomposition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of a compound of formula I or pharmaceutically acceptable saltthereof, and, if present, the second pharmaceutical formulation. Forexample, if the kit comprises a first composition comprising a compoundof formula I or pharmaceutically acceptable salt thereof and a secondpharmaceutical formulation, the kit may further comprise directions forthe simultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of formula I or pharmaceutically acceptablesalt thereof, such as tablets or capsules. Such a kit preferablyincludes a number of unit dosages. Such kits can include a card havingthe dosages oriented in the order of their intended use. An example ofsuch a kit is a “blister pack”. Blister packs are well known in thepackaging industry and are widely used for packaging pharmaceutical unitdosage forms. If desired, a memory aid can be provided, for example inthe form of numbers, letters, or other markings or with a calendarinsert, designating the days in the treatment schedule in which thedosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of formula I or pharmaceutically acceptable salt thereofcontained therein; and optionally (b) a second container with a secondpharmaceutical formulation contained therein, wherein the secondpharmaceutical formulation comprises a second compound withanti-hyperproliferative activity. Alternatively, or additionally, thekit may further comprise a third container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Where the kit comprises a composition of a compound of formula I orpharmaceutically acceptable salt thereof and a second therapeutic agent,i.e. the chemotherapeutic agent, the kit may comprise a container forcontaining the separate compositions such as a divided bottle or adivided foil packet, however, the separate compositions may also becontained within a single, undivided container. Typically, the kitcomprises directions for the administration of the separate components.The kit form is particularly advantageous when the separate componentsare preferably administered in different dosage forms (e.g., oral andparenteral), are administered at different dosage intervals, or whentitration of the individual components of the combination is desired bythe prescribing physician.

Specific Aspects of the Invention

In one specific aspect of the invention the hyperproliferative disorderis cancer.

In one specific aspect of the invention the cancer is associated withPTEN mutation.

In one specific aspect of the invention the cancer is associated withAKT mutation, overexpression or amplification.

In one specific aspect of the invention the cancer is associated withPI3K mutation.

In one specific aspect of the invention the cancer is selected from,breast, lung, ovarian, prostate (e.g., castration resistant prostatecancer), melanoma, gastric, colon, renal, head and neck, and giloma.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and 5-FU are administered tothe mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof, 5-FU, and oxaliplatin areadministered to the mammal and the cancer is gastric, ovarian, or colon.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof, 5-FU, and oxaliplatin areadministered to the mammal and the cancer is gastric, prostate, head orneck.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof, 5-FU, oxaliplatin, and folinicacid are administered to the mammal and the cancer is gastric, ovarian,or colon.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof, 5-FU, oxaliplatin, and folinicacid are administered to the mammal and the cancer is gastric, prostate,head or neck.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and carboplatin areadministered to the mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and carboplatin areadministered to the mammal and the cancer is breast, lung, or prostate.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and carboplatin areadministered to the mammal and the cancer is breast, lung, prostate,head or neck.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and irinotecan are administeredto the mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and irinotecan are administeredto the mammal and the cancer is colon.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof docetaxel are administered tothe mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and docetaxel are administeredto the mammal and the cancer is breast, giloma, lung, melanoma, ovarian,or prostate.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and docetaxel are administeredto the mammal and the cancer is breast, ovarian, or prostate.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and doxorubicin areadministered to the mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and doxorubicin areadministered to the mammal and the cancer is breast, lung, ovarian,giloma, or prostate.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and SN-38 are administered tothe mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and SN-38 are administered tothe mammal and the cancer is colon.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and temozolomide areadministered to the mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and temozolomide areadministered to the mammal and the cancer is giloma.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and a platinum agent areadministered to the mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and a platinum agent areadministered to the mammal and the cancer is ovarian.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and GDC-0973 or apharmaceutically acceptable salt thereof are administered to the mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and GDC-0973 or apharmaceutically acceptable salt thereof are administered to the mammaland the cancer is pancreatic, prostate, melanoma or breast.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and PLX-4032 or apharmaceutically acceptable salt thereof are administered to the mammal.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and PLX-4032 or apharmaceutically acceptable salt thereof are administered to the mammaland the cancer is melanoma.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof and erlotinib or apharmaceutically acceptable salt thereof are administered to the mammaland the cancer is non-small cell lung cancer.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof is administered orally.

In one specific aspect of the invention the compound of formula I or apharmaceutically acceptable salt thereof is formulated as a tablet.

General Preparative Procedures

EXAMPLES

In order to illustrate the invention, the following examples areincluded. However, it is to be understood that these examples do notlimit the invention and are only meant to suggest a method of practicingthe invention. Persons skilled in the art will recognize that thechemical reactions described may be readily adapted to prepare a numberof other AKT inhibitors of the invention, and alternative methods forpreparing the compounds of this invention are deemed to be within thescope of this invention. For example, the synthesis of non-exemplifiedcompounds according to the invention may be successfully performed bymodifications apparent to those skilled in the art, e.g., byappropriately protecting interfering groups, by utilizing other suitablereagents known in the art other than those described, and/or by makingroutine modifications of reaction conditions. Alternatively, otherreactions disclosed herein or known in the art will be recognized ashaving applicability for preparing other compounds of the invention.

Example 1

Preparation of(S)-3-amino-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-onedihydrochloride Step 1

To a 1 L round-bottom flask were added (R)-(+)-Pulegone (76.12 g, 0.5mmol), anhydrous NaHCO₃ (12.5 g) and anhydrous ether (500 mL). Thereaction mixture was cooled with ice-bath under nitrogen. The bromine(25.62 mL, 0.5 mmol) was added dropwise over 30 minutes. The mixture wasfiltered and carefully added to NaOEt (21%, 412 mL, 1.11 mmol) in anice-cooled bath. The mixture was stirred at room temperature overnightand then 1 L of 5% HCl and 300 mL of ether were added. The aqueous phasewas extracted with ether (2×300 mL). The combined organic phase waswashed with water, dried and concentrated. The residue was added to awarmed solution of semicarbazide hydrochloride (37.5 g) and NaOAc (37.5g) in water (300 mL), and then boiling ethanol (300 mL) was added togive a clear solution. The mixture was refluxed for 2.5 hours and thenstirred at room temperature overnight. The mixture was treated with 1 Lof water and 300 mL of ether. The aqueous phase was extracted with ether(2×300 mL). The combined organic phase was washed with water, dried andconcentrated. The residue was purified by vacuum distillation (73-76° C.at 0.8 mm Hg) to give (2R)-ethyl2-methyl-5-(propan-2-ylidene)cyclopentanecarboxylate (63 g, 64%). ¹H NMR(CDCl₃, 400 MHz) δ 4.13 (m, 2H), 3.38 (d, J=16 Hz, 0.5H), 2.93 (m,0.5H), 2.50-2.17 (m, 2H), 1.98 (m, 1H), 1.76 (m, 1H), 1.23 (m, 6H), 1.05(m, 6H).

Step 2

(2R)-Ethyl 2-methyl-5-(propan-2-ylidene)cyclopentanecarboxylate (24 g,0.122 mol) in ethyl acetate (100 mL) was cooled to −68° C. with dryice/isopropanol. Ozonized oxygen (5-7 ft³h⁻¹ of O₂) was bubbled throughthe solution for 3.5 hours. The reaction mixture was flushed withnitrogen at room temperature until the color disappeared. The ethylacetate was removed under vacuum and the residue was dissolved in 150 mLof acetic acid and cooled by ice water, and zinc powder (45 g) wasadded. The solution was stirred for 30 minutes and then filtered. Thefiltrate was neutralized with 2N NaOH (1.3 L) and NaHCO₃. The aqueousphase was extracted with ether (3×200 mL). The organic phase wascombined, washed with water, dried and concentrated to afford (2R)-ethyl2-methyl-5-oxocyclopentanecarboxylate (20 g, 96%). ¹H NMR (CDCl₃, 400MHz) δ 4.21 (m, 2H), 2.77 (d, J=11.2 Hz, 1H), 2.60 (m, 1H), 2.50-2.10(m, 3H), 1.42 (m, 1H), 1.33 (m, 3H), 1.23 (m, 3H).

Step 3

To a solution of a mixture of (2R)-ethyl2-methyl-5-oxocyclopentanecarboxylate (20 g, 117.5 mmol) and thiourea(9.2 g, 120.9 mmol) in ethanol (100 mL) was added KOH (8.3 g, 147.9mmol) in water (60 mL). The mixture was refluxed for 10 hours. Aftercooling, the solvent was removed and the residue was neutralized withconcentrated HCl (12 mL) at 0° C. and then extracted with DCM (3×150mL). The solvent was removed and the residue was purified by silica gelchromatography, eluting with Hexane/ethyl acetate (2:1) to give(R)-2-mercapto-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol (12g, 56%). MS (APCI+) [M+H]+ 183.

Step 4

To a suspension of(R)-2-mercapto-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol (12g, 65.8 mmol) in distilled water (100 mL) was added Raney Nickel (15 g)and NH4OH (20 mL). The mixture was refluxed for 3 hours then filtered,and the filtrate was concentrated to afford(R)-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol (9.89 g, 99%).MS (APCI+) [M+H]+ 151.

Step 5

A mixture of (R)-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol(5.8 g, 38.62 mmol) in POCl3 (20 mL) was refluxed for 5 minutes. ExcessPOCl3 was removed under vacuum and the residue was dissolved in DCM (50mL). The mixture was then added to saturated NaHCO3 (200 mL). Theaqueous phase was extracted with DCM (3×100 mL), and the combinedorganic phases were dried and concentrated. The residue was purified bysilica gel chromatography, eluting with ethyl acetate to give(R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine (3.18 g,49%). 1H NMR (CDCl3, 400 MHz) δ 8.81 (s, 1H), 3.47 (m, 1H), 3.20 (m,1H), 3.05 (m, 1H), 2.41 (m, 1H), 1.86 (m, 3H), 1.47 (m, 3H).

Step 6

To a solution of(R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine (2.5 g,14.8 mmol) in CHCl3 (60 mL) was added MCPBA (8.30 g, 37.0 mmol) in threeportions. The mixture was stirred at room temperature for 2 days. Themixture was cooled to 0° C. and to this was added dropwise Na2S2O3 (10g) in water (60 mL), followed by Na2CO3 (6 g) in water (20 mL). Thereaction mixture was stirred for 20 minutes. The aqueous phase wasextracted with CHCl3 (2×200 mL), and the combined organic phases wereconcentrated at low temperature (<25° C.). The residue was purified bysilica gel chromatography, eluting with ethyl acetate-DCM/MeOH (20:1) togive (R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-oxide(1.45 g, 53%). 1H NMR (CDCl3, 400 MHz) δ □8.66 (s, 1H), 3.50 (m, 1H),3.20 (m, 2H), 2.44 (m, 1H), 1.90 (m, 1H), 1.37 (d, J=7.2 Hz, 3H).

Step 7

A solution of(R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-oxide (1.45g, 7.85 mmol) in acetic anhydride (20 mL) was heated to 110° C. for 2hours. After cooling, excess solvent was removed under vacuum. Theresidue was purified by silica gel chromatography, eluting withHexane/ethyl acetate (3:1) to give(5R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-7-ylacetate (1.25 g, 70%). 1H NMR (CDCl3, 400 MHz) δ 8.92 (m, 1H), 6.30-6.03(m, 1H), 3.60-3.30 (m, 1H), 2.84 (m, 1H), 2.40-2.20 (m, 1H), 2.15 (d,J=6 Hz, 2H), 1.75 (m, 2H), 1.47 (d, J=6.8, 2H), 1.38 (d, J=7.2, 1H). MS(APCI+) [M+H]+ 227.

Step 8

To a solution of(5R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-7-ylacetate (0.5 g, 2.2 mmol) in NMP (10 mL) was added 1-Boc-piperazine (0.9g, 4.8 mmol). The reaction mixture was heated to 110° C. for 12 hours.After cooling, the reaction mixture was diluted with ethyl acetate (200mL) and washed with water (6×100 mL). The organic phase was dried andconcentrated. The residue was purified by silica gel chromatography,eluting with ethyl acetate to give tert-butyl4-((5R)-7-acetoxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(0.6 g, 72%). 1H NMR (CDCl3, 400 MHz) δ 8.60 (d, 1H), 6.05-5.90 (m, 1H),3.80-3.30 (m, 9H), 2.84 (m, 1H), 2.20-(m, 1H), 1.49 (s, 9H), 1.29-1.20(m, 3H). MS (APCI+) [M+H]+ 377. The resulting mixture of thediastereomers was purified by chiral separation HPLC (Chiralcel ODHcolumn, 250×20 mm, Hexane/EtOH 60:40, 21 mL/min). The first peak(RT=3.73 min) gave the tert-butyl4-((5R,7R)-7-acetoxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(0.144 g, 24%). The second peak (RT=5.66 min) gave the tert-butyl4-((5R,7S)-7-acetoxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(0.172 g, 29%). MS (APCI+) [M+H]+ 377.

Step 9

To a solution of tert-butyl4-((5R,7R)-7-acetoxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(0.144 g, 0.383 mmol) in THF (4 mL) was added LiOH (3M, 2 mL). Themixture was stirred at room temperature for 6 hours and then quenchedwith 2N HCl (3 mL). The solvent was removed and the residue was purifiedby silica gel chromatography, eluting with ethyl acetate to givetert-butyl4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(89 mg, 70%). %). 1H NMR (CDCl3, 400 MHz) δ 8.52 (s, 1H), 5.48 (br, 1H),5.14 (m, 1H), 3.82-3.40 (m, 9H), 2.20 (m, 2H), 1.49 (s, 9H), 1.19 (d,J=6.8 Hz, 3H). MS (APCI+) [M+H]+ 335.

Step 10

tert-Butyl4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylatewas treated with HCl (4M in dioxane, 2 mL) in DCM (5 mL) for 6 hours togive(5R,7R)-5-methyl-4-(piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-7-oldihydrochloride. MS (APCI+) [M+H]+ 235.

Step 11

Tert-butyl 2,4-dimethoxybenzylcarbamate (3.96 g, 14.8 mmol) wasdissolved in THF (74 mL) and cooled to −78° C. The solution was treatedwith butyl lithium (7.44 mL, 16.3 mmol) dropwise over a five minuteperiod to afford a pale-yellow solution. The solution was allowed tostir for 15 minutes before the chloro(methoxy)methane (1.35 mL, 17.8mmol) was added dropwise (neat). The reaction was stirred at −78° C. for10 minutes, then allowed to warm slowly to ambient temperatureovernight. The reaction was concentrated in vacuo to afford a yellow gelwhich was partitioned between half-saturated NH4Cl solution and ether.The aqueous layer was extracted once, and the organics were combined.The organic layer was washed with water, then brine, separated, driedover Na2SO4, filtered, and concentrated in vacuo. 1H NMR supports thedesired near-pure (>90%) tert-butyl2,4-dimethoxybenzyl(methoxymethyl)carbamate (4.81 g, 104% yield) as apale-yellow oil which was used without purification.

Step 12

(R)-4-benzyl-3-(2-(4-chlorophenyl)acetyl)oxazolidin-2-one (3.00 g, 9.10mmol) was dissolved in DCM (91 mL) and cooled to −78° C. A 1M toluenesolution of TiCl4 (11.4 mL, 11.4 mmol) was added to the solutionfollowed by DIEA (1.66 mL, 9.55 mmol) to afford a dark purple reaction.This was allowed to stir for 15 minutes before the tert-butyl2,4-dimethoxybenzyl(methoxymethyl)carbamate (3.40 g, 10.9 mmol) wasadded as a solution in DCM (10 mL) dropwise. The reaction was allowed tostir for 15 minutes at −78° C., then allowed to warm to −18° C. in abrine-ice bath for one hour. This reaction was allowed to warm slowly to0° C. over a 2.5 hour period. The reaction was then quenched with theaddition of saturated NH4Cl solution (100 mL). The layers wereseparated, and the organic layers was extracted once with DCM. Thecombined organic layers were dried over MgSO4, filtered, andconcentrated in vacuo to afford a yellow oil. The residue was purifiedby chromatography (silica gel eluted with 4:1 hexanes:ethyl acetate) toafford the pure material as a colorless oil tert-butyl2,4-dimethoxybenzyl((S)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl)carbamate(4.07 g, 73.5% yield). This tert-butyl2,4-dimethoxybenzyl((S)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl)carbamate(680 mg, 1.12 mmol) was dissolved in DCM (10.6 mL) and water (560 uL;19:1 DCM:water) at ambient temperature. The solution was treated withDDQ (380 mg, 1.67 mmol), and the reaction was allowed to stir for oneday to afford reaction completion by TLC and LCMS analysis. The reactionwas diluted with DCM and washed twice with half saturated NaHCO3solution. The organic layer was dried over MgSO4, filtered, andconcentrated in vacuo to afford a yellow-orange oil. The residue waspurified by chromatography (silica gel eluted with 9:1 hexanes:ethylacetate) to afford a mixture of the aldehyde by-product andtert-butyl(S)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropylcarbamate(not separable) as a pale-yellow oil (729 mg combined mass). LC/MS(APCI+) m/z 359.1 [M-BOC+H]+.

Step 13

35% H2O2 (0.240 mL, 2.91 mmol) was added to a solution of LiOH—H2O(0.0978 g, 2.33 mmol) in 2:1 THF:H2O (33 mL). The reaction mixture wasstirred at room temperature for 35 minutes, and then cooled to 0° C. Asolution containing a mixture oftert-butyl(S)-3-((R)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropylcarbamate(0.535 g, 1.17 mmol) and 2,4-dimethoxybenzaldehyde (0.194 g, 1.17 mmol)in THF (7 mL) was added dropwise by addition funnel. The ice bath wasallowed to slowly warm, and the reaction mixture was stirred overnight.The reaction mixture was then cooled to 0° C., and 1M Na2SO3 (7 mL) wasadded. The mixture was stirred for 5 minutes, and then warmed to roomtemperature and stirred an additional 20 minutes. The reaction mixturewas then transferred to a separatory funnel and washed with ether (3×).The aqueous layer was acidified with KHSO4(s), and the mixture wasextracted with DCM (2×). The combined extracts were dried (Na2SO4),filtered, and concentrated to give(S)-3-(tert-butoxycarbonylamino)-2-(4-chlorophenyl)propanoic acid (0.329g, 94.2% yield) as a white residue. LC/MS (APCI+) m/z 200 [M-BOC+H]+.

Step 14

4M HCl/dioxane (5.49 ml, 22.0 mmol) was added to a solution of(S)-3-(tert-butoxycarbonylamino)-2-(4-chlorophenyl)propanoic acid (0.329g, 1.10 mmol) in 2:1 dioxane:DCM (10 mL). The reaction mixture wasstirred at room temperature overnight (16 hours), after which it wasconcentrated to ⅓ volume. The resulting cloudy mixture was diluted withether, and the mixture was concentrated again to ⅓ volume. The mixturewas diluted again with ether (20 mL), and the solids were isolated byfiltration through a medium frit funnel with nitrogen pressure, rinsedwith ether (5×10 mL), dried under nitrogen pressure, and dried in vacuoto give (S)-3-amino-2-(4-chlorophenyl)propanoic acid hydrochloride(0.199 g, 76.8% yield) as a white powder. HPLC>99 area % pure. LC/MS(APCI+) m/z 200.

Step 15

Boc2O (0.368 g, 1.69 mmol) was added to a solution of(S)-3-amino-2-(4-chlorophenyl)propanoic acid hydrochloride (0.199 g,0.843 mmol) and tetramethylammonium hydroxide pentahydrate (0.382 g,2.11 mmol) in 10:1 MeCN:H2O (7.7 mL). The reaction mixture was stirredovernight at room temperature (12 hours), after which the MeCN wasremoved on a rotary evaporator. The mixture was diluted with water andwashed with ether (2×). The aqeuous layer was acidified with KHSO4(s),the mixture was extracted with DCM, and the combined extracts were dried(Na2SO4), filtered, and concentrated to give(S)-3-(tert-butoxycarbonylamino)-2-(4-chlorophenyl)propanoic acid (0.229g, 90.6% yield) as a foam. HPLC>99 area % pure. LC/MS (APCI+) m/z 200[M-BOC+H]+.

Step 16

To a solution of(5R,7R)-5-methyl-4-(piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-7-oldihydrochloride (88 mg, 0.29 mmol) and(S)-3-(tert-butoxycarbonylamino)-2-(4-chlorophenyl)propanoic acid (86mg, 0.29 mmol) in DCM (10 mL) and Diisopropylethylamine (0.22 mL, 1.3mmol) was added HBTU (110 mg, 0.29 mmol). The reaction mixture wasstirred at room temperature for 1 hour. The solvent was removed and theresidue was dissolved in ethyl acetate (100 mL), washed with water (6×50ml). The organic phase was dried and concentrated to givetert-butyl(S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropylcarbamate(116 mg, 78%). 1H NMR (CDCl3, 400 MHz) δ 8.51 (s, 1H), 7.34-7.20 (m,4H), 5.15-5.09 (m, 2H), 4.15-4.05 (m, 1H), 3.87-3.85 (m, 2H), 3.78-3.38(m, 7H), 3.22-3.19 (m, 1H), 2.20-2.10 (m, 2H), 1.48 (s, 9H), 1.41 (s,9H), 1.14-1.12 (d, J=7.2 Hz, 3H). MS (APCI+) [M+H]+ 516.

Step 17

Treatment oftert-butyl(S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropylcarbamatewith HCl (4M in dioxane, 2 mL) in DCM (5 mL) for 6 hours to give(S)-3-amino-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-onedihydrochloride. 1H NMR (D20, 400 MHz) δ 8.38 (s, 1H), 7.37-7.35 (d,J=8.4 Hz, 2H), 7.23-7.21 (d, J=8.4 Hz, 2H), 5.29-5.25 (m, 1H), 4.64 (s,9H), 4.31-4.28 (m, 1H), 4.11 (m, 1H), 3.88-3.79 (m, 2H), 3.70-3.20 (m,10H), 2.23-2.17 (m, 1H), 2.07-1.99 (m, 1H), 1.22-1.20 (m, 2H), 0.98-0.96(d, J=6.8 Hz, 2H). MS (APCI+) [M+H]+ 416.

Example 2

(S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-oneStep 1

Ethyl pulegenate (130 g, 662 mmol) in EtOAc (900 mL) was cooled to −78°C. using a dry ice-isopropanol bath. This mixture was subjected toozonolysis until the reaction turned purple in color. At this point,ozone generation ceased, and the reaction was removed from the dry-icebath. Oxygen was bubbled through the reaction mixture until it turnedyellow. The reaction mixture was concentrated under vacuum, and theresulting residue was dissolved in glacial acetic acid (400 mL). Thesolution was cooled to 0° C., and Zn dust (65 g, 993 mmol) was addedportionwise over 30 minutes. The reaction was then allowed to stir for 2hours, at which point the reaction mixture was filtered through a pad ofcelite to remove the zinc dust. The acetic acid was neutralized to pH 7with aqueous NaOH and NaHCO₃ and extracted with ether (3×800 mL). Thecombined organics were dried with brine, MgSO₄ and concentrated to give(2R)-ethyl 2-methyl-5-oxocyclopentane-carboxylate as a brown liquid (107g, 95%).

Step 2

Ammonium acetate (240.03 g, 3113.9 mmol) was added to a solution of(R)-ethyl 2-methyl-5-oxocyclopentanecarboxylate (106.0 g, 622.78 mmol)in MeOH (1.2 L). The reaction mixture was stirred at room temperatureunder nitrogen for 20 hours, after which it was complete as judged byTLC and HPLC. The reaction mixture was concentrated to remove MeOH. Theresulting residue was dissolved in DCM, washed twice with H₂O, once withbrine, dried (Na₂SO₄), filtered, and concentrated to give (R)-ethyl2-amino-5-methylcyclopent-1-enecarboxylate (102 g, 97% yield) as anorange oil. LC/MS (APCI+) m/z 170 [M+H]+.

Step 3

A solution containing (R)-ethyl2-amino-5-methylcyclopent-1-enecarboxylate (161.61 g, 955.024 mmol) andammonium formate (90.3298 g, 1432.54 mmol) in formamide (303.456 ml,7640.19 mmol) was heated to an internal temperature of 150° C. andstirred for 17 hours. The reaction mixture was cooled, and transferredto a 2 L single neck flask. Then excess formamidine was removed by highvacuum distillation. Once formamidine stopped coming over, the remainingoil in the still pot was dissolved in DCM and washed with brine (3×200mL). The combined aqueous washes were extracted with DCM. The combinedorganic extracts were dried (Na₂SO₄), filtered, and concentrated. Theresulting brown oil was dissolved in minimal DCM, and this solution wasadded using a separatory funnel to a stirred solution of ether (ca. 5vol of ether vs. DCM solution), causing some brown precipitate to form.This brown precipitate was removed by filtration through a medium fritfunnel which was rinsed with ether and disposed. The filtrate wasconcentrated, the trituration from ether repeated two more times andthen dried on high vacuum line to give(R)-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol (93.225 g,65.00% yield) as a brown-yellow pasty solid. LC/MS (APCI−) m/z 149.2.

Step 4

Neat POCl₃ (463.9 ml, 5067 mmol) was added slowly by addition funnel toa 0° C. solution of(R)-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol (152.2 g, 1013mmol) in DCE (1.2 L). After the addition was complete, the reactionmixture was warmed to room temperature, then heated to reflux andstirred for 70 minutes. The reaction was complete as determined by HPLC.The reaction mixture was cooled to room temperature, and the excessPOCl₃ was quenched in 4 portions as follows: Reaction mixturetransferred to separatory funnel and dripped into a beaker containingice and saturated NaHCO₃ solution cooled in an ice bath. Once theaddition of each portion of the reaction mixture was completed, thequenched mixture was stirred 30 minutes to ensure complete destructionof POCl₃ prior to transfer to separatory funnel. The mixture wastransferred to the separatory funnel and extracted twice with DCM. Thecombined extracts were dried (Na₂SO₄), filtered, and concentrated. Thecrude was purified on silica gel as follows: silica gel (1 kg) wasslurried in 9:1 hexane:ethyl acetate onto a 3 L fritted funnel, silicasettled under vacuum, topped with sand. The crude was loaded with aDCM/hexane mixture, and the compound was eluted using 1 L sidearm flasksunder vacuum. High Rf byproducts eluted first, then(R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine (104.4 g,61.09% yield) as a brown oil. Triethylamine (93.0 ml, 534 mmol) andtert-butyl piperazine-1-carboxylate (34.8 g, 187 mmol) was added to asolution of (R)-4-chloro-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine(30.0 g, 178 mmol) in n-BuOH (250 mL). The reaction mixture was heatedto reflux under nitrogen and stirred overnight (17 hours), after whichit was concentrated on a rotavap. The resulting oil was dissolved inDCM, washed with H₂O, dried (Na₂SO₄), filtered, and was concentrated.The resulting brown oil was purified on silica gel eluting first with2:1 hexanes:ethyl acetate until product eluting cleanly, then gradient1:1 to 1:5 DCM:ethyl acetate to give (R)-tertbutyl4-(5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(42.0 g, 74.1% yield) as a beige powder. LC/MS (APCI+) m/z 319.1 [M+H]⁺.

Step 5

Solid 77% max. MCPBA (23.9 g, 107 mmol) was added portionwise to a 0° C.solution of (R)-tert-butyl4-(5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(20.0 g, 62.8 mmol) in CHCl₃ (310 mL). The reaction mixture was stirred5 for minutes, then warmed to room temperature and stirred for 90minutes. HPLC looked similar after 7.5 hours. The reaction mixture wascooled to 0° C., then NaHCO₃ (13.2 g, 157 mmol) and another 0.5equivalents of m-CPBA were added. The reaction mixture was stirredovernight (14 hours). The reaction mixture was cooled to 0° C., and asolution of Na₂S₂O₃ (29.8 g, 188 mmol) in H₂O (50 mL) was added dropwiseby addition funnel. This was followed by a solution of Na₂CO₃ (24.6 g,232 mmol) in H₂O (70 mL) by addition funnel (mixture turns homogeneous).The reaction mixture was stirred for 30 minutes, then the mixture wasextracted with CHCl₃ (3×150 mL). The combined extracts were dried(Na₂SO₄), filtered, and concentrated to give the N-oxide. LC/MS (APCI+)m/z 335.1 [M+H]+.

Step 6

Ac₂O (77.0 ml, 816 mmol) was added to the N-oxide (21.0 g, 62.8 mmol)from Step 5. The reaction mixture was heated under nitrogen in a 90° C.sand bath and stirred for 100 minutes. The reaction mixture was cooledto room temperature, and excess acetic anhydride was removed by rotaryevaporation. The resulting oil was dissolved in DCM, which was thenpoured carefully into ice saturated Na₂CO₃. The mixture was extractedwith DCM, and the combined extracts were dried (Na₂SO₄), filtered, andconcentrated to give (5R)-tert-butyl4-(7-acetoxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(23.6 g, 100%) as a brown foam. LC/MS (APCI+) m/z 377.1 [M+H]+.

Step 7

LiOH—H₂O (6.577 g, 156.7 mmol) was added to a 0° C. solution of(5R)-tert-butyl4-(7-acetoxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(23.6 g, 62.69 mmol) in 2:1 THF:H₂O (320 mL). The reaction mixture wasstirred for 10 minutes, and then warmed to room temperature. LC/MSlooked the same at 3 hours and 4.5 hours. The reaction mixture wascooled to 0° C., and then saturated NH₄Cl was added to the mixture. Themixture was stirred for 5 minutes, and most of the THF was removed byrotary evaporation. The mixture was extracted with EtOAc (3×250 mL), andthe combined extracts were dried (Na₂SO₄), filtered, and concentrated.The crude was flashed on Biotage 65M: 4:1 DCM:ethyl acetate, thengradient to 1:1 to 1:4 DCM:ethyl acetate. Once the product was eluting,then ethyl acetate was flushed through the column. Then 30:1 DCM:MeOHeluted the rest of the product (8.83 g). The mixed fractions werere-flashed with Biotage 40M using the same conditions to give another2.99 g which gave a combined yield of (5R)-tert-butyl4-(7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(11.82 g, 56.38% yield) as a brown foam. LC/MS (APCI+) m/z 335.1 [M+H]+.

Step 8

A solution of DMSO (5.45 ml, 76.8 mmol) in DCM (50 mL) was addeddropwise by addition funnel to a −78° C. solution of oxalyl chloride(3.35 ml, 38.4 mmol) in DCM (150 mL). The reaction mixture was stirredfor 35 minutes, and then a solution of (5R)-tert-butyl4-(7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(9.17 g, 27.4 mmol) in DCM (80 mL) was added slowly by addition funnel.The reaction mixture was stirred another 1 hour at −78° C., after whichneat triethylamine (18.0 ml, 129 mmol) was added to the mixture. Thereaction mixture was then allowed to warm to room temperature, and thenit was stirred for 30 minutes. H₂O was added. The mixture was extractedwith DCM (3×200 mL), and the combined extracts were dried (Na₂SO₄),filtered, and concentrated in vacuo. The crude was purified on silicagel (Biotage 65M): the column was flushed with ca. 800 mL 4:1 DCM:EtOAc,then gradient to 1:1 DCM:ethyl acetate until product eluting, then 1:4DCM:EtOAc eluted product to give (R)-tert-butyl4-(5-methyl-7-oxo-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(7.5 g, 82.3% yield) as a brown foam. The foam was concentrated (3×)from DCM/hexanes, which gave a very light brown foam. HPLC>95% area.LC/MS (APCI+) m/z 333 [M+H]+.

Step 9

Triethylamine (4.33 ml, 31.1 mmol; degassed with nitrogen 30 minutesprior to use) and formic acid (1.36 ml, 36.1 mmol; degassed withnitrogen 30 minutes prior to use) were added to a solution of(R)-tert-butyl4-(5-methyl-7-oxo-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(9.75 g, 29.3 mmol) in DCM (210 mL; degassed with nitrogen 30 minutesprior to use). The mixture was stirred for 5 minutes, then a Ru catalyst(0.0933 g, 0.147 mmol) was added. The reaction was stirred underpositive nitrogen pressure overnight (18 hours). The reaction mixturewas concentrated to dryness and dried on high vacuum. The impurematerial was flashed on Biotage 65M loaded 1:1 DCM:ethyl acetate 500 mLflushed, then 1:4 DCM:ethyl acetate until product (2nd spot), thengradient to neat ethyl acetate, then 25:1 DCM:MeOH eluted rest ofproduct. The fractions were combined and concentrated on a rotaryevaporator. The residue was concentrated again from DCM/hexanes to givea mixture of tert-butyl4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(major) and tert-butyl4-((5R,7S)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(minor) (9.35 g, 95.3% yield) as a beige foam. LC/MS (APCI+) m/z 335[M+H]+. 1H NMR (CDCl3) shows 88% de by integration of carbinol methine.

Step 10: 4-Nitrobenzoyl chloride (4.27 g, 23.0 mmol) was added to a 0°C. solution of tert-butyl4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(7.0 g, 20.9 mmol) and triethylamine (4.38 ml, 31.4 mmol) in DCM (110mL). The reaction mixture was stirred at room temperature overnight,after which saturated NaHCO₃ was added. The mixture was stirred 10minutes, and then extracted with DCM. The combined extracts were dried(Na₂SO₄), filtered, and concentrated. The crude was flashed on Biotage65M (3:1 hexanes:ethyl acetate loaded crude, then 2:1 hexanes:ethylacetate eluted tert-butyl4-((5R,7R)-5-methyl-7-(4-nitrobenzoyloxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylateand a few mixed fractions). Then tert-butyl4-((5R,7S)-5-methyl-7-(4-nitrobenzoyloxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylatewas eluted using 1:2 hexanes:ethyl acetate. The fractions with productwere concentrated by rotary evaporation to give tert-butyl4-((5R,7R)-5-methyl-7-(4-nitrobenzoyloxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(8.55 g, 84.5% yield) as a yellow foam. LC/MS (APCI+) m/z 484 [M+H]+. 1HNMR (CDCl3) shows single diastereomer). The fractions with otherdiastereomer were concentrated by rotary evaporation to give tert-butyl4-((5R,7S)-5-methyl-7-(4-nitrobenzoyloxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(0.356 g, 3.52% yield) as a brown foam. LC/MS (APCI+) m/z 484 [M+H]+.

Step 11

LiOH—H₂O (0.499 g, 11.9 mmol) was added to a 0° C. solution oftert-butyl4-((5R,7R)-5-methyl-7-(4-nitrobenzoyloxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(2.30 g, 4.76 mmol) in 2:1 THF:H₂O (40 mL). The reaction mixture waswarmed to room temperature and stirred for 1 hour. The THF was removedby rotary evaporation, saturated NaHCO₃ was added, and the mixture wasextracted with ethyl acetate. The combined extracts were washed (1×)with saturated NaHCO₃, dried (Na₂SO₄), filtered, and concentrated togive tert-butyl4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(1.59 g, 100.0% yield) as a yellow foam. HPLC after workup justproduct>98 area % pure. LC/MS (APCI+) m/z 335 [M+H]+. The tert-butyl4-((5R,7S)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylatewas prepared using an analogous method.

Step 12

4M HCl/dioxane (11.2 ml, 44.9 mmol) was added to a solution oftert-butyl4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazine-1-carboxylate(0.600 g, 1.79 mmol) in dioxane (15 mL). The reaction mixture wasstirred at room temperature under nitrogen overnight (20 hours). Themixture was concentrated to dryness and dried on high vacuum line. Thecrude was suspended in ether, sonicated, and stirred for 5 minutes. Thesolids were isolated by filtration through a medium fit funnel withnitrogen pressure, rinsed with ether, dried under nitrogen pressure, anddried further on a hi vacuum line to give(5R,7R)-5-methyl-4-(piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-7-oldihydrochloride (0.440 g, 79.8% yield) as a yellow powder. LC/MS (APCI+)m/z 235. The(5R,7S)-5-methyl-4-(piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-7-oldihydrochloride was prepared using an analogous method.

Step 13

Methyl 2-(4-chlorophenyl)acetate (36.7 g, 199 mmol) and paraformaldehyde(6.27 g, 209 mmol) were dissolved/suspended in DMSO (400 mL) and treatedwith NaOMe (537 mg, 9.94 mmol). The mixture was allowed to stir at roomtemperature for 2 hours to completion by TLC analysis of the crude. Thereaction was poured into ice-cold water (700 mL; white emulsion) andneutralized with the addition of 1M HCl solution. The aqueous layer wasextracted with ethyl acetate (3×), and the organics were combined. Theorganic layer was washed with water (2×), brine (1×), separated, driedover MgSO₄, filtered, and concentrated in vacuo to afford the crudeproduct as a yellow oil. The residue was loaded onto a large frittedfiltered with silica gel and eluted with 9:1 hexanes:ethyl acetate untilthe starting material/olefin were collected. The plug was then elutedwith 1:1 hexanes:ethyl acetate until the pure desired product was elutedcompletely. The concentrated pure fractions yielded methyl2-(4-chlorophenyl)-3-hydroxypropanoate as a colorless oil (39.4 g, 92%).

Step 14

Methyl 2-(4-chlorophenyl)-3-hydroxypropanoate (39.4 g, 184 mmol) wasdissolved in DCM (500 mL) and treated with TEA (64.0 mL, 459 mmol). Thesolution was cooled to 0° C. and slowly treated with MsCl (15.6 mL, 202mmol), then allowed to stir for 30 minutes to completion by TLCanalysis. The solution was partitioned with 1N HCl solution, and theaqueous layer was extracted once with DCM. The combined organic layerwas washed once more with 1N HCl solution, separated, washed withdiluted NaHCO₃ solution, and separated. The organic layer was dried overMgSO₄, filtered, and concentrated in vacuo to afford an orange oil. Theresidue was loaded onto a large fritted filter with a plug of silica geland eluted with 9:1 hexanes:ethyl acetate affording the pure desiredproduct by TLC analysis. The concentrated pure fractions yielded themethyl 2-(4-chlorophenyl)acrylate as a colorless oil (30.8 g, 85%). Thismethyl 2-(4-chlorophenyl)acrylate (500 mg, 2.54 mmol) was added as asolution in THF (1.35 mL) to a stirring solution of i-PrNH₂ (217 uL,2.54 mmol) in THF (5.0 mL) at 0° C. The reaction was allowed to stir atroom temperature overnight to completion by LCMS analysis. The Boc2O(584 uL, 2.54 mmol) was added to the stirring amine via pipet. Thereaction was allowed to stir overnight to completion by LCMS and TLCanalysis of the mixture. The solution was concentrated in vacuo toafford methyl3-(tert-butoxycarbonyl(isopropyl)amino)-2-(4-chlorophenyl)propanoate asa colorless oil (854 mg, 94%). LC/MS (APCI+) m/z 256.1 [M-Boc]+.

Step 15

Methyl3-(tert-butoxycarbonyl)isopropyl)amino)-2-(4-chlorophenyl)propanoate(133 g, 374 mmol) was dissolved in THF (1.0 L) and treated with KOTMS(56.0 g, 392 mmol) at room temperature. The mixture was allowed to stirovernight to completion by LCMS analysis of the crude. The mixture wasconcentrated in vacuo to afford a wet foam, which was allowed to dryunder vacuum overnight to afford potassium3-(tert-butoxycarbonyl(isopropyl)amino)-2-(4-chlorophenyl)propanoate asa white solid (148.7 g, 105%). LC/MS (APCI+) m/z 242.1 [M-Boc-K]+.

Step 16

Potassium3-(tert-butoxycarbonyl(isopropyl)amino)-2-(4-chlorophenyl)propanoate(77.2 g, 203 mmol) was dissolved in THF (515 mL) and treated withpivaloyl chloride (26.3 mL, 213 mmol) at room temperature. The mixturewas allowed to stir for 3 hours to form the mixed anhydride.(S)-4-benzyloxazolidin-2-one (46.1 g, 260 mmol) was dissolved in THF(600 mL) and cooled to −78° C. in a separate flask. The solution wastreated with n-BuLi (102 mL of a 2.50M solution in hexanes, 254 mmol)and allowed to stir for one hour. The prepared anhydride solution wasadded to the stirring Li-oxazolidinone via cannula, and the mixture wasallowed to warm to room temperature overnight. The mixture was quenchedwith the addition of saturated ammonium chloride solution, thenpartitioned between more water and ethyl acetate. The aqueous layer wasextracted several times, and the organics were combined. The organiclayer was washed with water, then brine, separated, dried over MgSO₄,filtered, and concentrated in vacuo. The residue was purified/separated(diastereomers) via chromatography (silica gel eluted with 4:1hexanes:ethyl acetate) to afford the completely separated diastereomersas viscous oils:tert-butyl(R)-3-((S)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl(isopropyl)carbamate(12.16 g, 24% based on ½ of acid racemate) andtert-butyl(S)-3-((S)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl(isopropyl)carbamate(39.14 g, 77% based on ½ of acid racemate). LC/MS (APCI+) m/z 401.2[M-Boc]+.

Step 17

LiOH—H₂O (168 mg, 4.00 mmol) was added to a stirring solution of THF (30mL) and water (15 mL) at room temperature until it was dissolved. Themixture was treated with hydrogen peroxide (658 uL of a 35% wt. solutionin water, 8.00 mmol) and allowed to stir at room temperature for 10minutes. The reaction was cooled to 0° C. in an ice bath, and thetert-butyl(S)-3-((S)-4-benzyl-2-oxooxazolidin-3-yl)-2-(4-chlorophenyl)-3-oxopropyl(isopropyl)carbamate(1.00 g, 2.00 mmol) was added dropwise via addition funnel as a solutionin THF (15 mL) over a 10 minutes. The mixture was allowed to stirovernight at room temperature to completion by LCMS analysis of thecrude. The reaction was cooled to 0° C., and then treated with 1M Na₂SO₃(9.00 mL) solution via addition funnel over a ten minute period. Afterthe addition was complete, the mixture was allowed to warm to roomtemperature for 10 minutes. The mixture was concentrated to remove theTHF, and then diluted with water. The aqueous layer was washed twicewith ethyl acetate (discarded). The aqueous layer was partitioned withethyl acetate, then treated dropwise while stirring with 1M HCl until pH2-3 was attained. The aqueous layer was extracted twice with ethylacetate, and the organics were combined. The organic was washed withbrine, separated, dried over MgSO₄, filtered, and concentrated in vacuo.The colorless oil product was dried under high vacuum for one hour toafford(S)-3-(tert-butoxycarbonyl(isopropyl)amino)-2-(4-chlorophenyl)propanoicacid as a viscous oil/foam (685 mg, 100%). LC/MS (APCI+) m/z 242.1[M-Boc]+.

Step 18

A solution of(5R,7R)-5-methyl-4-(piperazin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidin-7-oldihydrochloride (2.92 g, 9.51 mmol) and(S)-3-(tert-butoxycarbonyl)isopropyl)amino)-2-(4-chlorophenyl)propanoicacid (3.25 g, 9.51 mmol) in DCM (40 mL) and DIEA (5.0 mL, 28.7 mmol) wasstirred at room temperature for 10 minutes. HBTU (3.61 g, 9.51 mmol) wasadded to the mixture. The mixture was stirred at room temperature for 1hour. The solvent was removed, and the residue was dissolved in ethylacetate (500 mL) and washed with water (6×100 mL). The organic phase wasdried and concentrated. The residue was subject to columnchromatography, eluted by EtOAc-DCM/MeOH (20:1) to givetert-butyl(S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl(isopropyl)carbamate(3.68 g, 69%.) LC/MS (APCI+) m/z 558.2 [M+H]+.

Step 19

Thetert-butyl(S)-2-(4-chlorophenyl)-3-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-oxopropyl(isopropyl)carbamate (2.50 g, 4.48 mmol) was dissolved in dioxane (22.4 mL) andtreated with 4M HCl in dioxane (22.4 mL, 89.6 mmol) at room temperature.The resulting solution was allowed to stir overnight to completion byLCMS analysis of the crude. The solution was concentrated in vacuo toafford a gel that was dissolved in a minimal amount of methanol (10 mL).The solution was transferred via pipette to stirred ether (300 mL) toafford a white precipitate of desired product. The addition was abouthalf when the white precipitate melted into a yellow gel. The materialwas concentrated in vacuo to afford a yellow gel which was allowed tostand under reduced pressure overnight to yield(S)-2-(4-chlorophenyl)-1-(4-05R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-onedihydrochloride as a light yellow powder (2.14 g, 90%).

¹H NMR (D₂O, 400 MHz□□ δ 8.39 (s, 1H), 7.37-7.35 (d, J=8.4 Hz, 2H),7.23-7.20 (d, J=8.4 Hz, 2H), 5.29-5.25 (m, 1H), 4.33-4.29 (m, 1H),4.14-4.10 (m, 1H), 3.89-3.19 (m, 11H), 2.23-2.17 (m, 1H), 2.08-1.99 (m,1H), 1.20-1.18 (m, 6H), 0.98-0.96 (d, J=6.8 Hz, 3H). MS (APCI+) [M+H]⁺458.

Examples 3-9 shown in Table 1 can also be made according to theabove-described methods.

TABLE 1 LCMS or ¹H Example Structure Name NMR 3

(S)-2-(4-chlorophenyl)-3- (dimethylamino)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H- cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one 444.1 4

(S)-2-(3-fluoro-4- (trifluoromethyl)phenyl)-1-(4-((5R,7S)-7-hydroxy-5-methyl-6,7- dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3- (isopropylamino)propan-1-one 510.3 5

(S)-2-(4-chlorophenyl)-1-(4- ((5R,7S)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin- 4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one 458.3 6

(R)-2-(4-chlorophenyl)-1-(4- ((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin- 4-yl)piperazin-1-yl)-3-(isopropylamino)propan-1-one 458 7

(S)-2-(4-chloro-3- fluorophenyl)-3- (cyclopropylmethylamino)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7- dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)propan-1-one LCMS (APCI+) m/z 488, 490 [M + H]+ 8

(S)-2-(4-chloro-3- fluorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H- cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(tetrahydro-2H- pyran-4-ylamino)propan-1-one LCMS(APCI+) m/z 518, 520 [M + H]+ 9

(S)-2-(4-chloro-3- fluorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H- cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-((1r,4S)-4- methoxycyclohexylamino)propan-1- oneLCMS (APCI+) m/z 546

Example 10

(S)-2-(4-cyclopropylphenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-2-((S)-pyrrolidin-2-yl)ethanoneStep 1

Cyclopropylmagnesium bromide (64.0 mL, 32.00 mmol) in THF was treatedwith a solution of zinc (II) chloride (64.00 mL, 32.00 mmol) in THF. Themixture was stirred at ambient temperature for 20 minutes.2-(4-Bromophenyl)acetonitrile (5.228 g, 26.67 mmol) and bis[tri-t-butylphosphine]palladium (0.6814 g, 1.333 mmol) were added as a solution inTHF (2 mL). The reaction was stirred at ambient temperature undernitrogen for 12 hours. The reaction was quenched with saturated NH4Cl,diluted with methylene chloride and separated. The aqueous layer waswashed with methylene chloride (2×), and then the combined organiclayers were washed with water (3×), dried over Na2SO4 and concentratedin vacuo. The crude product was subjected to chromatography on SiO2eluting with 25:1 hexanes/ethyl acetate to yield2-(4-cyclopropylphenyl)acetonitrile (2.76 g, 66%). 1H NMR (CDCl3, 400MHz) □ 7.20 (d, J=8.2, 2H), 7.07 (d, J=8.2, 2H), 3.70 (s, 2H), 1.94-1.85(m, 1H), 1.01-0.95 (m, 2H), 0.71-0.66 (m, 2H).

Step 2

Methanol (65 mL) was cooled to 0° C. and saturated with HCl (g). Thissolution was treated with a solution of2-(4-cyclopropylphenyl)acetonitrile (2.76 g, 17.56 mmol) in methanol (6mL). The reaction mixture was heated to reflux overnight under a dryingtube containing CaSO4. The reaction was cooled and concentrated invacuo. The crude mixture was re-suspended in ethyl acetate and water andthen separated. The organic layer was washed with saturated NaHCO3,saturated NaCl, dried over Na2SO4 and concentrated in vacuo to providemethyl 2-(4-cyclopropylphenyl)acetate as an oil (3.10 g, 93%). 1H NMR(CDCl3, 400 MHz) δ 7.16 (d, J=8.3, 2H), 7.02 (d, 2H), 3.68 (s, 3H), 3.58(s, 2H), 1.92-1.83 (m, 1H), 0.97-0.91 (m, 2H), 0.70-0.64 (m, 2H).

Step 3

Methyl 2-(4-cyclopropylphenyl)acetate (3.10 g, 16.30 mmol) was dissolvedin a mixture of THF/MeOH/water (2:2:1, 80 mL), and the solution wastreated with lithium hydroxide hydrate (0.8548 g, 20.37 mmol). Themixture was then stirred at ambient temperature for 4 hours. Thereaction mixture was neutralized to a pH of 4 with 3N HCl andconcentrated in vacuo. The solids were re-dissolved in ethyl acetate andwater. The pH was re-adjusted to a pH of about 3 to about 4 with 3N HCl.The layers were then separated. The aqueous layer was washed with ethylacetate (2×). The combined organic layers were then washed withsaturated NaCl, dried over Na2SO4 and concentrated to yield2-(4-cyclopropylphenyl)acetic acid (2.82 g, 98%). 1H NMR (CDCl3, 400MHz) □ 7.16 (d, J=8.2, 2H), 7.03 (d, 2H), 3.60 (s, 2H), 1.92-1.83 (m,1H), 098-0.91 (m, 2H), 0.70-0.64 (m, 2H).

Step 4: 2-(4-Cyclopropylphenyl)acetic acid (2.82 g, 16.003 mmol) wascombined with (R)-4-benzyloxazolidin-2-one (3.4030 g, 19.204 mmol) intoluene (14 mL). The suspension was treated with triethylamine (6.6917mL, 48.010 mmol) and then heated to 80° C. The solution was treateddropwise with a solution of pivaloyl chloride (1.9893 mL, 16.003 mmol)in toluene (3.5 mL). The reaction was heated overnight at 80° C. Thereaction was cooled and washed with 2N HCl and then separated. Theaqueous layer was washed with toluene, and the combined organics werethen washed with 2N HCl, water, saturated NaHCO3 (2×), saturated NaCl,dried over Na2SO4 and concentrated in vacuo. The crude product wassubjected to chromatography on SiO2 eluting with 9:1 hexanes/ethylacetate to yield(R)-4-benzyl-3-(2-(4-cyclopropylphenyl)acetyl)oxazolidin-2-one (3.43 g,64%). 1H NMR (CDCl3, 400 MHz) □ 7.33-7.20 (m, 5H), 7.16-7.11 (m, 2H),7.05 (d, J=8.2, 2H), 4.70-4.63 (m, 1H), 4.32-4.14 (m, 4H), 3.26 (dd,J1=3.2, J2=13.3, 1H), 2.75 (dd, J1=9.5, J2=13.3, 1H), 1.93-1.85 (m, 1H),0.98-0.92 (m, 2H), 0.72-0.66 (m, 2H).

Step 5

(S)-2-((S)-1-(tert-Butoxycarbonyl)pyrrolidin-2-yl)-2-(4-cyclopropylphenyl)aceticacid was prepared according to the procedure described for Example 1,using (R)-4-benzyl-3-(2-(4-cyclopropylphenyl)acetyl)oxazolidin-2-one(0.287 g, 26%). MS (ESI+) [M+H] 345.7.

Step 6

(S)-tert-Butyl2-((S)-1-(4-cyclopropylphenyl)-2-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-2-oxoethyl)pyrrolidine-1-carboxylatewas prepared according to the procedure described for Example 3 using(S)-2-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-2-(4-cyclopropylphenyl)aceticacid, (0.199 g, 94%). MS (ESI+) [M+H] 562.1.

Step 7

(S)-2-(4-Cyclopropylphenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-2-4S)-pyrrolidin-2-yl)ethanonewas prepared according to the procedure described for Example 3 using(S)-tert-butyl2-((S)-1-(4-cyclopropylphenyl)-2-(445R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-2-oxoethyl)pyrrolidine-1-carboxylate(0.145 g, 77%). MS (ESI+) [M+H] 462.2. 1H NMR (CD3OD, 400 MHz) □ 8.56(s, 1H), 7.26 (d, 2H), 7.13 (d, 2H), 5.29 (dd, 1H), 5.32-5.26 (dd, 1H),4.32 (d, 1H), 4.29-4.18 (m, 1H), 4.12-3.95 (m, 2H), 3.88-3.61 (m, 6H),3.51-3.38 (m, 1H), 3.35-3.30 (m, 1H), 2.32-2.24 (m, 1H), 2.22-2.03 (m,2H), 1.95-1.85 (m, 2H), 1.82-1.73 (m, 2H), 1.40-1.34 (m, 1H), 1.16 (d,3H), 1.01-0.95 (m, 2H), 0.69-0.64 (m, 2H).

Examples shown in Table 2 can also be made according to the abovedescribed methods.

TABLE 2 Example Structure Name LCMS or ¹H NMR 11

4-((S)-2-(4-((5R,7R)-7-hydroxy-5- methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4- yl)piperazin-1-yl)-1-((S)-1-methylpyrrolidin-2-yl)-2- oxoethyl)benzonitrile m/z 461.3; ¹H NMR (500MHz, DMSO-D6) d ppm 8.65 (s, 1H), 7.85 (d, 2H), 7.65 (d, 2H), 5.10 (t,1H), 4.80 (d, 1H), 4.10-3.85 (m, 5H), 3.68 (m, 2H), 3.40 (m, 2H), 2.90(s, 3H), 2.20-2.02 (m, 2H), 1.93 (m, 2H), 1.68 (m, 1H), 1.50 (m, 1H),1.35-1.25 (m, 11H), 1.10 (d, 3H) 12

(S)-1-(4-((5R,7R)-7-hydroxy-5- methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4- yl)piperazin-1-yl)-2-((S)-pyrrolidin-2-yl)-2-(4- (trifluoromethyl)phenyl)ethanone m/z 490.3; ¹H NMR (500 MHz,DMSO-D6) d ppm 9.18 (m, 1H), 8.85 (m, 1H), 8.57 (s, 1H), 7.78 (d, 2H),7.62 (d, 2H), 5.04 (t, 1H), 4.48 (d, 1H), 4.02 (m, 2H), 3.95 (m, 2H),3.75-3.50 (m, 6H), 3.42 (m, 2H), 3.30-3.10 (m, 4H), 2.10-1.90 (m 3H),1.75 (m, 1H), 1.70-1.50 (m, 2H), 1.04 (d, 3H) 13

(S)-2-(4-chloro-3-fluorophenyl)-2- ((S)-5,5-dimethylpyrrolidin-2-yl)-1-(4-((5R,7R)-7-hydroxy-5-methyl- 6,7-dihydro-5H-cyclopenta[d]pyrimidin-4- yl)piperazin-1-yl)ethanone LCMS (apci+) 502[M + H]+; 2.68 min; HPLC r.t. = 1.98 min, >97% purity; ¹H NMR (400 MHz,D₂O) d ppm 8.37 (s, 1H), 7.43 (t, J = 8.2 Hz, 1H), 7.16 (d, J = 9.8 Hz,1H), 7.06 (d, J = 8.2 Hz, 1H), 5.24 (t, J = 7.8 Hz, 1H), 4.27 (d, J =9.4 Hz, 1H), 4.22-4.02 (m, 1H), 3.88-3.75 (m, 2H), 3.72-3.60 (m, 1H),3.59-3.41 (m, 4H0, 3.37-3.22 (m, 1H), 2.24-2.11 (m, 0.5H), 2.10-1.94 (m,0.5H), 1.89-1.71 (m, 4H), 1.36 (s, 3H), 1.30 (s, 3H), 0.96 (d, J = 7.0Hz, 3H)

Example 14 In Vitro Cell Proliferation Assays

The in vitro potency of the combinations of the compound of Example 2with certain specific chemotherapeutic agents was measured using theCellTiter-Glo® Luminescent Cell Viability Assay, commercially availablefrom Promega Corp., Madison, Wis. This homogeneous assay method is basedon the recombinant expression of Coleoptera luciferase (U.S. Pat. No.5,583,024; U.S. Pat. No. 5,674,713; U.S. Pat. No. 5,700,670) anddetermines the number of viable cells in culture based on quantitationof the ATP present, an indicator of metabolically active cells (Crouchet al (1993) J. Immunol. Meth. 160:81-88; U.S. Pat. No. 6,602,677). TheCellTiter-Glo® Assay was conducted in 96 or 384 well format, making itamenable to automated high-throughput screening (HTS) (Cree et al (1995)AntiCancer Drugs 6:398-404). The homogeneous assay procedure involvesadding the single reagent (CellTiter-Glo® Reagent) directly to cellscultured in serum-supplemented medium. Cell washing, removal of mediumand multiple pipetting steps are not required. The system detects as fewas 15 cells/well in a 384-well format in 10 minutes after adding reagentand mixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g., 96 or 384 well format. Data can be recorded byluminometer or CCD camera imaging device. The luminescence output ispresented as relative light units (RLU), measured over time.

The anti-proliferative effects of combinations of the compound ofExample 2 and certain chemotherapeutic agents were measured using theCellTiter-Glo® Assay. EC₅₀ values were established for the testedcompounds and combinations. The range of in vitro cell potencyactivities was about 100 nM to about 10 μM.

Example 15 In Vivo Tumor Xenograft Efficacy

The efficacy of representative combinations of the invention may bemeasured in vivo by implanting allografts or xenografts of cancer cellsin rodents and treating the tumor-bearing animals with the combinations.Variable results are to be expected depending on the cell line, thepresence or absence of certain mutations in the tumor cells, thesequence of administration the compound of Example 2 andchemotherapeutic agent, dosing regimen, and other factors. Subject micewere treated with drug(s) or control (Vehicle) and monitored overseveral weeks or more to measure the time to tumor doubling, log cellkill, and tumor inhibition.

Results for representative combinations of the invention that weretested in this model are presented in the Figures.

The data in Figures demonstrates that representative combinationsprovide improved results compared to the administration of therespective agents individually.

It has been determined that certain combinations of the inventionprovide improved effects against certain cancer phenotypes. For example,certain combinations of the invention provide improved effects againstcancers associated with PTEN mutation, AKT mutation (e.g.,overexpression or amplification), PI3K mutation, or Her2/ErbB2amplification. Accordingly, certain combinations described herein may beparticularly useful against these types of cancers. For example, ingastric cancer, PTEN-loss predicts better efficacy with certaincombinations of the invention (e.g., a compound of formula I with5-FU/cisplatin), and in prostate cancer a stronger effect was seen for acombination of a compound of formula I and docetaxel in PTEN-null lines.

PTEN null status may be measured by any suitable means as is known inthe art. In one example, IHC is used. Alternatively, Western blotanalysis can be used. Antibodies to PTEN are commercially available(Cell Signaling Technology, Beverly, Mass., Cascade Biosciences,Winchester, Mass.). Example procedures for IHC and Western blot analysisfor PTEN status are described in Neshat, M. S. et al. Enhancedsensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR, Proc.Natl Acad. Sci. USA 98, 10314-10319 (2001) and Perren, A., et. al.Immunohistochemical Evidence of Loss of PTEN Expression in PrimaryDuctal Adenocarcinomas of the Breast, American Journal of Pathology,Vol. 155, No. 4, October 1999. Additionally, cancers associated with AKTmutation, PI3K mutation, and with Her2/ErbB2 amplification can beidentified using techniques that are known in the art.

EGFR mutations can be detected by using commercially available kits. Forexample, the Cobas® EGFR Mutation Test (Roche Molecular Diagnostics) canbe used. The kit identifies mutations in exons 18, 19, 20 and 21 of theEGFR gene. The Cobas® EGFR Mutation Test offers broad mutation coverageof EGFR exons 18, 19, 20 and 21 to help select cancer patients, e.g.,non-small cell lung cancer (NSCLC) patients, for treatment with EGFRinhibitors, such as erlotinib, and in one embodiment, in combinationwith formula I compounds, e.g., GDC-0068.

In one specific aspect, the invention provides a method for treating apatient having a cancer that is associated with PTEN mutation or loss ofexpression, AKT mutation or amplification, PI3K mutation oramplification, or Her2/ErbB2 amplification comprising administering acombination of the invention to the patient. In another aspect, theinvention provides a method for identifying a patient having a cancerthat that can be treated with a combination of the invention comprisingdetermining if the patient's cancer is associated with PTEN mutation orloss of expression, AKT mutation or amplification, PI3K mutation oramplification, or Her2/ErbB2 amplification, wherein association of thepatient's cancer with PTEN mutation or loss of expression, AKT mutationor amplification, PI3K mutation or amplification, or Her2/ErbB2amplification is indicative of a cancer that can be treated with acombination of the invention. In a further aspect, the inventionprovides a method further comprising treating the patient so identifiedwith a combination of the invention. Further, since numerousmodifications and changes will be readily apparent to those skilled inthe art, it is not desired to limit the invention to the exactconstruction and process shown as described above. Accordingly, allsuitable modifications and equivalents may be considered to fall withinthe scope as defined by the claims that follow.

We claim:
 1. A compound of formula Ia:

or a pharmaceutically acceptable salt thereof, for therapeutic use forimproving the quality of life of a patient treated for ahyperproliferative disorder with erlotinib or a pharmaceuticallyacceptable salt thereof, wherein the hyperproliferative disorder isnon-small cell lung cancer or pancreatic cancer.
 2. A method fortreating a hyperproliferative disorder in a mammal comprising,administering to the mammal a combination of, a) a compound of FormulaIa:

or a pharmaceutically acceptable salt thereof; and b) erlotinib or apharmaceutically acceptable salt thereof, wherein the hyperproliferativedisorder is non-small cell lung cancer or pancreatic cancer.
 3. Themethod of claim 2 wherein the non-small cell lung cancer or pancreaticcancer is associated with PTEN mutation.
 4. The method of claim 2wherein the non-small cell lung cancer or pancreatic cancer isassociated with AKT mutation, overexpression or amplification.
 5. Themethod of claim 2 wherein the non-small cell lung cancer or pancreaticcancer is associated with PI3K mutation.
 6. The method of claim 2wherein the non-small cell lung cancer or pancreatic cancer isassociated with Her2/ErbB2 amplification.
 7. The method of claim 2wherein the combination provides a synergistic effect in treating thehyperproliferative disorder.
 8. The method of claim 7 whereinCombination Index value of the synergistic effect is less than about0.8.
 9. The method of claim 2, wherein a) a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof; and b) erlotinib or apharmaceutically acceptable salt thereof are administered separately.10. The method of claim 2, wherein a) a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof; and b) erlotinib or apharmaceutically acceptable salt thereof are administered sequentially.11. The method of claim 2, wherein a) a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof; and b) erlotinib or apharmaceutically acceptable salt thereof are administeredsimultaneously.
 12. A kit comprising a compound of formula Ia:

or a pharmaceutically acceptable salt thereof, erlotinib or apharmaceutically acceptable salt thereof, a container, and a packageinsert or label indicating the administration of the compound of formulaIa with erlotinib or a pharmaceutically acceptable salt thereof fortreating a hyperproliferative disorder, wherein the hyperproliferativedisorder is non-small cell lung cancer or pancreatic cancer.
 13. Aproduct comprising a compound having formula Ia:

or a pharmaceutically acceptable salt thereof, and erlotinib or apharmaceutically acceptable salt thereof as a combined preparation forseparate, simultaneous or sequential use in the treatment of ahyperproliferative disorder, wherein the hyperproliferative disorder isnon-small cell lung cancer or pancreatic cancer.